Nascent osteoblast matrix inhibits osteogenesis of human mesenchymal stem cells in vitro

Abstract

Introduction: Adult mesenchymal stem cells (MSCs) are considered promising candidates for cell-based therapies. Their potential utility derives primarily from their immunomodulatory activity, multi-lineage differentiation potential, and likely progenitor cell function in wound healing and repair of connective tissues. However, in vitro, MSCs often senesce and spontaneously differentiate into osteoblasts after prolonged expansion, likely because of lack of regulatory microenvironmental signals. In vivo, osteoblasts that line the endosteal bone marrow surface are in close proximity to MSCs in the marrow stroma and thus may help to regulate MSC fate.

Methods: We examined here how osteogenic differentiation of MSCs in vitro is affected by exposure to osteoblastic cells (OBCs). Human bone marrow MSCs were exposed to OBCs, derived by induced osteogenic differentiation of MSCs, either directly in contact co-cultures, or indirectly to OBC-conditioned medium or decellularized OBC extracellular matrix (ECM).

Results: Our results showed that OBCs can act as negative regulators of MSC osteogenesis. mRNA expression profiling revealed that OBCs did not affect MSC osteogenesis in direct contact cultures or via secreted factors. However, seeding MSCs on decellularized OBC ECM significantly decreased expression of several osteogenic genes and maintained their fibroblastic morphologies. Proteomic analysis identified some of the candidate protein regulators of MSC osteogenesis.

Conclusions: These findings provide the basis for future studies to elucidate the signaling mechanisms responsible for osteoblast matrix-mediated regulation of MSC osteogenesis and to better manipulate MSC fate in vitro to minimize their spontaneous differentiation.

Fig. 1

Lysis of osteoblastic cells. MSC cultures were osteogenically differentiated for 15 days, lysed with DOC or water, and examined microscopically. a-c Phase contrast images of residual matrix after lysis. d-f Fluorescent live (green)/dead (red) merged images. g-i AlzR staining of matrix calcification. j-l blue staining of ALP enzymatic activity. (a, d, g, j) OBCs before lysis. (b, e, h, k) OBCs after DOC lysis. (c, f, i, l) OBCs after water lysis. Microscopy images taken at 10×. MSC mesenchymal stem cells, AlzR Alizarin Red S, ALP alkaline phosphatase, OBCs osteoblastic cells

Fig. 2

Water-treated OBC matrix enhances matrix mineralization of MSCs. OBC matrix was prepared from the cells shown in Fig. 1 at day 15 of osteogenic induction. Cultures were stained with AlzR to detect mineralization. MSCs are shown on plastic at day 12 of culture in EM (d) and OM (a). e DOC- and (f) water-treated matrices, without MSCs on top and maintained in OM for 12 days, show that water-treated OBC matrix sometimes remineralizes on its own (f). b and c show MSCs seeded on DOC- and water-treated OBC matrix, respectively, after 12 days in OM. DOC-matrix did not affect mineralization compared to cells on plastic (a). While water-treated matrix alone (f) showed a high level of AlzR staining even in the absence of live cells, the presence of MSCs enhanced staining (c). OBC osteoblastic cells, MSC mesenchymal stem cells, AlzR Alizarin Red S, EM expansion medium, OM osteogenic medium

Fig. 3

OBC matrix slows MSC proliferation in EM and OM. MSCs were plated on plastic or DOC-treated or water-treated OBC matrix and maintained in OM or EM. Their proliferation was examined on culture days 6 and 12. a In OM, MSC proliferation was reduced by 2-fold on DOC-treated OBC matrix and 1.2-fold on water-treated matrix. b MSC proliferation on plastic was 1.3-fold lower in OM than in EM, but this reduced proliferation is absent when comparing cells in EM and OM on water-treated OBC matrix. Fold changes are compared to the pre-culture value and expressed as mean ± SEM. Data are from a single experiment, representative of three experiments. *, p < 0.5, **, p < 0.01, ***, p < 0.001. OBC osteoblastic cells, MSC mesenchymal stem cells, EM expansion medium, OM osteogenic medium, SEM standard error of the mean

Fig. 4

OBC matrix suppresses MSC expression of osteogenic genes. MSCs were seeded on top of OBC matrix (treated with water or DOC) and cultured in OM for 6 and 12 days. Gene expression of a OC, Runx2, b ALP, and ColI was assayed by real-time RT-PCR. Expression levels were normalized to GAPDH and shown as a percentage of the maximum level within each experiment. Values are means ± SEM of five experiments. Asterisks indicate significance between experimental conditions and the OM control. Bars show significance between matrix conditions. #, p < 0.1, *, p < 0.05, **, p < 0.01, ***, p < 0.001. OBC osteoblastic cells, MSC mesenchymal stem cells, OM osteogenic medium, RT-PCR reverse transcription-polymerase chain reaction, SEM standard error of the mean

Fig. 5

OBC matrix preserves the naïve, fibroblastic morphology of MSCs. Fluorescence microscopy of Calcein AM-labeled cells shows that, even at day 12, MSCs cultured in OM and seeded on OBC matrices (c, DOC- and d, water-treated) retain their fibroblastic morphology, like MSCs cultured in EM on plastic (a), instead of becoming cuboidal, like cells cultured in OM on plastic (b). a, b Viewed at day 6. OBC osteoblastic cells, MSC mesenchymal stem cells, OM osteogenic medium, EM expansion medium

Fig. 6

2D-DIGE analysis of OBC matrix proteins. a A fluorescent image of a representative 2D gel. Blue bands at left represent fluorescent M_r standards. DOC-treated OBC matrix proteins were labeled with Cy3 (green); water-lysed OBC matrix proteins with Cy5 (red). The linear, immobilized IEF gradient gel is at the top with the acidic end (pH 3) at the left and the basic end (pH 10) at the right. The white box shows the approximate area of the Coomassie-stained gel shown in (b) from which the protein spots were excised. Excised spots are circled in pink and labeled with a spot number. 2D-DIGE 2D-differential in-gel electrophoresis, OBC osteoblastic cells, IEF isoelectric focusing gel

Fig. 7

Graphs showing the relative abundance of matrix proteins from different spots. a Spot 449. b Spot 1096

Fig. 8

Connections to regulators of osteogenesis and cellular proliferation exhibited by proteins identified by 2D-DIGE. This network, generated by Ingenuity®, portrays relationships described in the literature between all of the proteins identified by 2D-DIGE. Runx2 was added to emphasize the participation of these proteins in the osteogenic pathway. The proteins from the gel spots are colored in varying shades of red relative to the match score they received from Mascot. Together with TGF-β1, Runx2 and TP53 are hubs for regulation of osteogenesis and cellular proliferation. (Note: Gray arrows indicate action, not up/down-regulation. Only lines leading to and away from the four focus genes are color-coded.) Protein symbols are listed in (Additional file 8: Table S2). 2D-DIGE 2D-differential in-gel electrophoresis