Adipose stem cells (ASCs) are an attractive cell source for treating many human diseases including osteoporosis. However, the molecular mechanisms accounting for ASC osteogenesis are poorly known. In this study, ASCs were first isolated from the fat tissues from the patients with osteoporosis. The global transcriptome profile between osteogenic differentiated ASCs and undifferentiated ASCs was compared using RNA sequencing (RNA-seq). Then, bioinformatic analysis was performed to reveal the central genes and pathways that regulated the osteogenic differentiation of ASCs. One of the interested genes C5AR1 was chosen for further investigation. A total of 1521 upregulated and 3020 downregulated genes were identified between the ASCs with osteogenic induction and controls. Functional gene ontology analysis revealed that these significantly differentially expressed genes (DEGs) were associated with cell cycle, protein binding, and nucleotide binding. Pathway analysis showed that many canonical pathways, such as the MAPK signaling pathway and the PI3K-AKT pathway, might actively be involved in regulating osteogenic differentiation of ASCs. A total of three subnetworks and 20 central nodes were identified by the protein-protein interaction analysis. In addition, the expression level of C5AR1 was significantly increased during osteogenic differentiation of ASCs. The downregulation of C5AR1 dramatically reduced the expression levels of osteogenic differentiation biomarkers and calcium nodule formation capacity. Collectively, we have provided a number of novel genes and pathways that might be indispensable for ASC osteogenic differentiation. Manipulating the levels of this candidate gene might contribute to the osteoporosis therapy.
Osteoporosis is the most frequently occurring metabolic bone disease that affects many millions of people around the world. It is featured by low bone mineral density and microarchitectural deterioration of bone tissue, resulting in enhanced bone fragility and a consequent increase in fracture risk [
Bone marrow-derived mesenchymal stem cells (BMMSCs) are the most well-known and well-characterized source of adult stem cells [
In this study, we first aimed to determine the osteogenic potential of ASCs derived from patients with osteoporosis. Then, the key genes and pathways involved in the osteogenic differentiation of ASCs were explored by RNA sequencing and bioinformatic analysis. Finally, the role of interested gene C5AR1 in regulating ASC osteogenic differentiation was investigated.
Human ASCs were isolated from the deep and superficial layers of abdominal fat tissues of five donors with osteoporosis and two healthy controls. All donors signed a written informed consent for using their tissues, and the study was approved by the institutional Medical Ethics Committee of Changzhou Second People’s Hospital, Nanjing Medical University. Briefly, the adipose tissues were rinsed three times with sterile PBS to remove debris and red blood cells. Then, they were minced into small pieces (1-2 mm3) with surgical scissors. The small tissue pieces were digested in PBS containing 3 mg/mL collagenase type I (Sigma, St. Louis, MO, USA) at 37°C for 1 h. Single cell suspensions were obtained by passing the cells through a 70
Total RNA was extracted with the RNeasy plus mini kit (QIAGEN, Hilden, Germany) according to the manufacturer’s protocol. The RNA sequencing of control and osteogenic differentiated ASCs (each with 3 biological replicates) was performed using the BGISEQ-500 sequencing system by Beijing Genomics Institute (BGI), China. Briefly, the RNA was sheared and reverse transcribed to cDNA using random primers to obtain cDNA library. Raw sequencing reads were filtered to get clean reads. Bowtie2 and HISAT were used to map clean reads to reference gene and genome, respectively. Gene expression was quantified and normalized using the RSEM tool. The NOISeq method was used to screen out differentially expressed genes (DEGs) between two groups. The standard for the cutoff value was
Gene ontology (GO) enrichment analysis and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analysis were performed using the Cytoscape Bingo plugin and KEGG pathway database (
ASCs were transfected with double-stranded siRNA using the RNAiMAX transfection reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instruction. siRNAs of C5AR1 (siC5AR1) or scrambled control siRNAs (sicontrol) (Santa Cruz Biotechnology Inc., Dallas, TX, USA) were mixed with the transfection reagent, respectively, and then added to the cell culture.
Total RNA was isolated from cells sing Quick-RNA Miniprep (Zymo Research, Irvine, CA, USA) based on the manufacturer’s instructions. SuperScript III Reverse Transcriptase (Invitrogen, Carlsbad, CA, USA) was used to synthesize the first-strand cDNA. The PCR reaction was performed with Light Cycler 480@ SYBR Green I Master Mix (Roche Applied Science, Indianapolis, IN, USA) on the CFX96 Real-Time PCR detection system (Bio-Rad Laboratories Inc., Hercules, CA, USA). GAPDH was employed as the internal control, and the 2-
After 24 h of serum starvation, the cells were seeded into a 96-well plate at a density of 3000 cells/well. At the indicated time points, 20
The proteins extracted from the cell samples were separated on a 4%-12% Bis-Tris NuPAGE gel and transferred onto a polyvinylidene difluoride membrane using a Trans-blot SD semidry transfer cell (Bio-Rad, Hercules, CA, USA). The membranes were blocked with 5% nonfat milk for 1 h at room temperature and incubated with primary C5AR1 antibody (1 : 100; Santa Cruz Biotechnology) overnight, followed by horseradish peroxidase- (HRP-) linked secondary antibody (1 : 5000; GE Healthcare, Piscataway, NJ, USA). Signal detection was performed with the ECL-Plus western blotting reagent kit (GE Healthcare).
Human ASCs were seeded in 6-well plates at a density of
The data were expressed as the
The ASCs derived from the patients with osteoporosis mainly had round-up or spindle-shaped morphology, which were similar to the ASCs from healthy donors (Figure
The ASCs derived from the patients with osteoporosis mainly had round-up or spindle-shaped morphology, which were similar to the ASCs from healthy donors (a). MTT assay and alizarin red S staining showed that ASCs derived from the osteoporosis patients had similar proliferation and osteogenic differentiation capacity with those from healthy controls (b, c).
Global transcriptome profile of osteogenic differentiated ASCs and undifferentiated ASCs. (a) Volcano plot of the DEGs between osteogenic differentiated ASCs and undifferentiated ASCs. Totally, 1521 upregulated and 3020 downregulated genes were identified. (b) Heat map of the DEGs between osteogenic differentiated ASCs and undifferentiated ASCs. (c) KEGG pathway analysis of the DEGs between osteogenic differentiated ASCs and undifferentiated ASCs.
The KEGG analysis showed that the top enriched pathways included the PI3K signaling pathway, MAPK signaling pathway, cell cycle, regulation of actin cytoskeleton, and Rap1 signaling pathway (Figure
Gene ontology analyses of the significantly changed genes between osteogenic differentiated ASCs and the control cells according to their biological process, cellular component, and molecular function.
Figure
PPI network analysis of the most significant changed genes was constructed. A total of 3 subnetworks and 20 central nodes were identified by Cytoscape MCODE plugin.
Our qPCR data showed that the expression levels of SAA1, C5AR1, APOD, and CNR1 were significantly increased in osteogenic differentiated ASCs compared with the undifferentiated controls. On the contrary, the expression levels of ACAN, CHRM2, CD36, and CHRM2 were dramatically reduced in ASCs with osteogenic induction (Figure
qPCR validation of the RNA-seq data. Consistent with our RNA-seq data, the expression levels of SAA1, C5AR1, APOD, and CNR1 were significantly upregulated in osteogenic differentiated ASCs, while the levels of ACAN, CHRM2, CD36, and CHRM2 were remarkably downregulated.
The expression levels of C5AR1 mRNA and protein were gradually increased with osteogenic induction (Figure
The inhibition of C5AR1 suppressed the osteogenic differentiation of ASCs. (a) The expression of C5AR1 mRNA and protein was significantly increased during osteogenic differentiation of ASCs. (b) The expression level of C5AR1 mRNA and protein was dramatically downregulated following siC5AR1 transfection. (c) C5AR1 downregulation inhibited the expression of osteogenic differentiation markers. (d) C5AR1 downregulation suppressed the calcium nodule formation capacity of ASCs.
In this study, we have shown that the ASCs derived from patients with osteoporosis had strong osteogenic differentiation potential, which further supports the usage of ASCs as the seed cells for treating osteoporosis to avoid the potential graft rejection owing to allograft. In addition, many significantly upregulated or downregulated genes during osteogenic differentiation of ASCs were identified. Bioinformatic analysis revealed many key central genes and pathways that might play essential roles in regulating ASC osteogenic differentiation. To the best of our knowledge, many genes are reported to be potential regulators of ASC osteogenic differentiation for the first time. Moreover, the expression level of the central node gene C5AR1 was gradually increased during osteogenic differentiation of ASCs. The downregulation of C5AR1 significantly suppressed the osteogenic differentiation of ASCs, indicating that C5AR1 was a positive regulator of ASC osteogenic differentiation.
The KEGG pathway analysis revealed many important pathways that might be involved in regulating osteogenic differentiation of ASCs. For instance, the MAPK signaling pathway has been demonstrated to function as key players in skeletal development and bone homeostasis especially osteoblast commitment and differentiation [
We then used the most upregulated and downregulated genes to construct the PPI network. Our results identified many interested genes that might play an important role in regulating osteogenic differentiation of ASCs. For instance, recombined serum amyloid A (SAA) could induce the osteogenic differentiation of MSCs by regulating the TLR4 receptor [
C5AR1 is a G protein-coupled receptor for C5a and also an N-linked glycosylated protein [
In conclusion, based on our reliable transcriptome data, we have identified many novel genes and pathways that might be involved in regulating the osteogenic differentiation of ASCs. In addition, C5AR1 might act as a positive modulator for ASC osteogenic differentiation, indicating that manipulating the C5AR1 expression level might contribute to bone regeneration and tissue engineering.
The RNA-seq data used to support the findings of this study are included within the supplementary information file.
The authors declare that they have no conflicts of interest.
Jinluo Cheng and Xinyuan Zhao contributed equally to this work.
This study was supported by the Key Technology R&D Program of Changzhou City (CE20155051).
The significant differentially expressed genes between osteogenic differentiated ASCs and controls.