Tissue source determines the differentiation potentials of mesenchymal stem cells: a comparative study of human mesenchymal stem cells from bone marrow and adipose tissue

Abstract

Background: Mesenchymal stem cells (MSCs) possess intrinsic regeneration capacity as part of the repair process in response to injury, such as fracture or other tissue injury. Bone marrow and adipose tissue are the major sources of MSCs. However, which cell type is more effective and suitable for cell therapy remains to be answered. The intrinsic molecular mechanism supporting the assertion has also been lacking.

Methods: Human bone marrow-derived MSCs (BMSCs) and adipose tissue-derived MSCs (ATSCs) were isolated from bone marrow and adipose tissue obtained after total hip arthroplasty. ATSCs and BMSCs were incubated in standard growth medium. Trilineage differentiation including osteogenesis, adipogenesis, and chondrogenesis was performed by addition of relevant induction mediums. The expression levels of trilineage differentiation marker genes were evaluated by quantitative RT-PCR. The methylation status of CpG sites of Runx2, PPARγ, and Sox9 promoters were checked by bisulfite sequencing. In addition, ectopic bone formation and calvarial bone critical defect models were used to evaluate the bone regeneration ability of ATSCs and BMSCs in vivo.

Results: The results showed that BMSCs possessed stronger osteogenic and lower adipogenic differentiation potentials compared to ATSCs. There was no significant difference in the chondrogenic differentiation potential. The CpG sites of Runx2 promoter in BMSCs were hypomethylated, while in ATSCs they were hypermethylated. The CpG sites of PPARγ promoter in ATSCs were hypomethylated, while in BMSCs they were hypermethylated. The methylation status of Sox9 promoter in BMSCs was only slightly lower than that in ATSCs.

Conclusions: The epigenetic memory obtained from either bone marrow or adipose tissue favored MSC differentiation along an osteoblastic or adipocytic lineage. The methylation status of the main transcription factors controlling MSC fate contributes to the differential differentiation capacities of different source-derived MSCs.

Keywords: Adipose tissue-derived MSCs; Bone marrow-derived MSCs; Epigenetic regulation; Mesenchymal stem cells.

Fig. 1

Characterization of cell surface markers of ATSCs and BMSCs. Cell surface markers of ATSCs and BMSCs (both at passage 3) were analyzed using flow cytometry. Antibodies against CD90, CD44, CD73, CD31, and CD45 were used to characterize ATSCs and BMSCs. ATSC: adipose tissue-derived MSC, BMSC: bone marrow-derived MSC, MSC: mesenchymal stem cell

Fig. 2

Difference in osteogenesis ability between ATSCs and BMSCs in vitro. a–d Total RNA extracted from ATSCs and BMSCs or cells subjected to OIM for 7 days. Relative expression levels of ALP, Runx2, OPN, and OCN checked by qRT-PCR. β-actin as an internal control. Data expressed as mean ± SD (n = 3). *p < 0.05, compared with MSCs in OIM; #p < 0.05, compared with MSCs in α-MEM. e, f Alizarin Red S staining of calcium deposits formed by ATSCs and BMSCs. ATSCs and BMSCs cultured in OIM for 14 days, then cells fixed and stained with Alizarin Red S. ALP: alkaline phosphatase, ATSC: adipose tissue-derived MSC, BMSC: bone marrow-derived MSC, MEM: minimum essential medium, MSC: mesenchymal stem cell, OCN: Osteocalcin, OIM: osteogenic induction medium, OPN: Osteopontin, Runx2: runt-related transcription factor 2

Fig. 3

Difference of adipogenesis between ATSCs and BMSCs in vitro. a–d Total RNA extracted from ATSCs and BMSCs when cells were subjected to AIM for 7 days. Relative expression levels of PPARγ, CEBPα, AP2, and LPL checked by qRT-PCR. β-actin as an internal control. Data expressed as mean ± SD (n = 3). *p < 0.05, compared with MSCs in OIM; #p < 0.05, compared with MSCs in α-MEM. e, f ATSCs and BMSCs cultured in AIM for 21 days, then cells fixed and stained with Oil Red O. AP2 adipocyte protein 2, ATSC adipose tissue-derived MSC, CEBPα CCAAT/enhancer-binding protein alpha, BMSC bone marrow-derived MSC, LPL lipoprotein lipase, MEM minimum essential medium, MSC mesenchymal stem cell, OIM osteogenic induction medium, PPARγ peroxisome proliferator-activated receptor gamma

Fig. 4

DNA methylation status of Runx2 and PPARγ in ATSCs and BMSCs. DNA methylation status of Runx2 (a) and PPARγ (b) promoters in ATSCs and BMSCs were examed using sodium bisulfite sequencing. Top panel indicates CpG dinucleotide position of the Runx2 and PPARγ promoter regions. Each PCR product was subcloned and subjected to nucleotide sequencing analysis. Ten representative sequenced clones depicted by filled (methylated) and open (unmethylated) circles for each CpG site. ATSC: adipose tissue-derived MSC, BMSC: bone marrow-derived MSC, MSC: mesenchymal stem cell, PPARγ: peroxisome proliferator-activated receptor gamma, Runx2: runt-related transcription factor 2

Fig. 5

BMSCs showed slightly stronger chondrogenic differentiation potential. a, b Total RNA extracted from ATSCs and BMSCs when cells were subjected to CIM for 10 days. Relative expression levels of Sox9 and Col2 checked by qRT-PCR. β-actin as an internal control. Data expressed as mean ± SD. p < 0.05. c DNA methylation status of Sox9 promoter in ATSCs and BMSCs using sodium bisulfite sequencing. Top panel indicates CpG dinucleotide position of the Sox9 promoter region and numbers show positions of CpGs relative to the translation start site. Each PCR product was subcloned and subjected to nucleotide sequencing analysis. Ten representative sequenced clones depicted by filled (methylated) and open (unmethylated) circles for each CpG site. ATSC: adipose tissue-derived MSC, Col2: Collagen type II, BMSC: bone marrow-derived MSC, MSC: mesenchymal stem cell

Fig. 6

Ectopic bone formation of ATSCs and BMSCs in nude mice. a, ATSCs and BMSCs were loaded onto sterilized porous calcium phosphate restorable granules, then implanted subcutaneously into the dorsal surfaces of nude mice. Transplants were harvested 8 weeks later for histological examination. Sections stained with routine hematoxylin and eosin (HE) and immunohistochemical (IHC) staining with anti-OCN antibody. b, The bone formation area were measured and there were significantly more bone formation in the BMSC group, *p,<0.05. ATSC: adipose tissue-derived MSC, BMSC: bone marrow-derived MSC, MSC: mesenchymal stem cells, NB: new bone tissue, OCN: Osteocalcin

Fig. 7

ATSCs and BMSCs enhanced calvarial bone repair in nude mice. a X-ray and microCT analysis 3D reconstruction of calvarial bone samples. b MicroCT analysis showed the new bone volume in the BMSC group was significantly increased (*) compared to that of the control group. ATSC adipose tissue-derived MSC, BMSC bone marrow-derived MSC, CT computed tomography, MSC mesenchymal stem cell