Human circulating fibrocytes have the capacity to differentiate osteoblasts and chondrocytes

Abstract

Fibrocytes are bone marrow-derived cells. Fibrocytes can differentiate into adipocyte- and myofibroblast-like cells. Since fibrocytes can behave like mesenchymal progenitor cells, we hypothesized that fibrocytes have the potential to differentiate into other mesenchymal lineage cells, such as osteoblasts and chondrocytes. In this study, we found that fibrocytes differentiated into osteoblast-like cells when cultured in osteogenic media in a manner similar to osteoblast precursor cells. Under these conditions, fibrocytes and osteoblast precursor cells displayed increased calcium deposition, and increased expression of specific osteogenic genes. In addition, dephosphorylation of cAMP-responsive element binding protein was associated with the increased ratio of receptor activator of the NF-kappaB Ligand/osteoprotegerin gene expression and enhanced gene expression of osterix in these cells under these conditions. Both events are important in promoting osteogenesis. In contrast, fibrocytes and mesenchymal stem cells cultured in chondrogenic media in the presence of transforming growth factor-beta3 were found to differentiate to chondrocyte-like cells. Fibrocytes and mesenchymal stem cells under these conditions were found to express increased levels of aggrecan and type II collagen genes. Transcription factor genes associated with chondrogenesis were also found to be induced in fibrocytes and mesenchymal stem cells under these conditions. In contrast, beta-catenin protein and the core binding factor alpha1 subunit protein transcription factor were decreased in expression under these conditions. These data indicate that human fibrocytes have the capability to differentiate into osteoblast- and chondrocyte-like cells. These findings suggest that such cells could be used in cell-based tissue-regenerative therapy.

Fig. 1

Fibrocytes display the ability to differentiate into osteoblasts. (A) Time course of calcium deposition from fibrocytes undergoing differentiation to osteoblast-like cells in the presence of osteogenic media, as assessed by von Kossa staining. (B) von Kossa staining and (C), Alizarin Red S staining of fibrocytes and OBPC exposed to osteogenic media for 21 days. Magnification 400×. Representative picture from at least 5 experiments for each panel is shown.

Fig. 2

Fibrocytes undergoing osteogenesis express genes for osteogenic markers. (A) Time course of gene expression of osteonectin, osteopontin, and osteocalcin from fibrocytes and OBPC undergoing differentiation to osteoblast-like cells in the presence or absence of osteogenic media. GAPDH served as a control housekeeping gene. (B) Time course of osteonectin protein expression from fibrocytes and OBPC exposed to the presence or absence of osteogenic media. β-tubulin was used as a control for loading equivalent amounts of protein per well. Results are representative of 3 experiments. (C) Immunofluorescent image of osteonectin protein from fibrocytes and OBPC exposed to the presence or absence of osteogenic media. Magnification 400×. Results are representative of 2 experiments.

Fig. 3

Fibrocytes undergoing osteogenic differentiation demonstrate dephosphorylation of CREB protein, enhanced ratio of RANKL/OPG gene expression, and induction of osterix gene expression. (A) Dephosphorylation of CREB is seen in fibrocytes and OBPC undergoing differentiation to osteoblast-like cell in the presence of osteogenic media for 21 days. (B) Ratio of RANKL/OPG gene expression from fibrocytes exposed to the presence or absence of osteogenic media for 21 days. Intensity of the bands from RT-PCR with the corresponding genes was quantified by densitometer (Quantity-1 software, Bio-Rad). Representative image from 3 different experiments of gel electrophoresis is shown. N=6 for each data for quantification of band intensity using densitometer software (*p<0.01). (C) Osterix and Cbfa1 gene expression from fibrocytes and OBPC exposed to the presence or absence of osteogenic media for 21 days. Representative image from 3 different experiments of gel electrophoresis was shown.

Fig. 4

Fibrocytes display the ability to differentiate into chondrocytes. (A) Time course of alcian blue staining of fibrocytes exposed to chondrogenic media in the presence or absence of TGF-β3 (10 ng/ml). (B) Safranin-O staining of fibrocytes exposed to chondrogenic media with or without TGF-β3. Magnification 400×. Representative images from at least 3 experiments.

Fig. 5

Fibrocytes undergoing chondrogenesis express specific chondrogenic genes. (A) Time course of gene expression of aggrecan and Col2A1 from fibrocytes and MSC exposed to chondrogenic media with or without TGF-β3 (10 ng/ml). (B) Time course of aggrecan protein expression from fibrocytes and MSC exposed to chondrogenic media with or without TGF-β3 (10 ng/ml). Results are representative of 3 experiments.

Fig. 6

Chondrogenic transcription factor gene expression (i.e., SOX5, SOX6, and SOX9) are upregulated during fibrocyte differentiation to chondrocytes. (A) Expression of SOX9, SOX5, and SOX6 transcription factor genes from fibrocytes and MSC exposed to chondrogenic media with or without TGF-β3 (10 ng/ml) for 3 days. (B) Time course of the expression of SOX9 protein from fibrocytes and MSC exposed to chondrogenic media in the presence or absence of TGF-β3 (10 ng/ml). Results are representative of 3 experiments. (C) Expression of β-catenin, Cbfa1, and SOX9 genes from fibrocytes exposed to chondrogenic media for 3 days in the presence or absence of TGF-β3 (10 ng/ml). Results are representative of 3 experiments.