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Identification of Pathways Mediating Growth Differentiation Factor5-Induced Tenogenic Differentiation in Human Bone Marrow Stromal Cells.

Tan SL, Ahmad TS, Ng WM, Azlina AA, Azhar MM, Selvaratnam L, Kamarul T - PLoS ONE (2015)

Bottom Line: The genes identified as potentially associated with tenogenic differentiation were ARHGAP29, CCL2, integrin alpha 8 and neurofilament medium polypeptides.These genes, were mainly associated with cytoskeleton reorganization (stress fibers formation) signaling.Understanding and elucidating the tenogenic differentiation signalling pathways are important for future optimization of tenogenic hMSCs for functional tendon cell-based therapy and tissue engineering.

View Article: PubMed Central - PubMed

Affiliation: Tissue Engineering Group (TEG), National Orthopaedic Centre of Excellence for Research and Learning (NOCERAL), Department of Orthopaedic Surgery, Faculty of Medicine, University of Malaya, Pantai Valley, Kuala Lumpur, 50603, Malaysia.

ABSTRACT
To date, the molecular signalling mechanisms which regulate growth factors-induced MSCs tenogenic differentiation remain largely unknown. Therefore, a study to determine the global gene expression profile of tenogenic differentiation in human bone marrow stromal cells (hMSCs) using growth differentiation factor 5 (GDF5) was conducted. Microarray analyses were conducted on hMSCs cultures supplemented with 100 ng/ml of GDF5 and compared to undifferentiated hMSCs and adult tenocytes. Results of QuantiGene® Plex assay support the use and interpretation of the inferred gene expression profiles and pathways information. From the 27,216 genes assessed, 873 genes (3.21% of the overall human transcriptome) were significantly altered during the tenogenic differentiation process (corrected p<0.05). The genes identified as potentially associated with tenogenic differentiation were ARHGAP29, CCL2, integrin alpha 8 and neurofilament medium polypeptides. These genes, were mainly associated with cytoskeleton reorganization (stress fibers formation) signaling. Pathway analysis demonstrated the potential molecular pathways involved in tenogenic differentiation were: cytoskeleton reorganization related i.e. keratin filament signaling and activin A signaling; cell adhesion related i.e. chemokine and adhesion signaling; and extracellular matrix related i.e. arachidonic acid production signaling. Further investigation using atomic force microscopy and confocal laser scanning microscopy demonstrated apparent cytoskeleton reorganization in GDF5-induced hMSCs suggesting that cytoskeleton reorganization signaling is an important event involved in tenogenic differentiation. Besides, a reduced nucleostemin expression observed suggested a lower cell proliferation rate in hMSCs undergoing tenogenic differentiation. Understanding and elucidating the tenogenic differentiation signalling pathways are important for future optimization of tenogenic hMSCs for functional tendon cell-based therapy and tissue engineering.

No MeSH data available.


Related in: MedlinePlus

The candidate tenogenic markers (COL-I, TNMD, TNC and SCX) expression of GDF5 (100 ng/ml)-induced hMSC on day 4 (A, B, C) and day 10 (D, E, F) by immunofluorescence imaging.The extent of candidate tenogenic markers expressions were increased in GDF5 treated hMSC compared to the untreated control. An increase in the intensity of the expression of these markers was also observed in day 10 GDF5-induced hMSCs compared to that of day 4. Images were captured at 63X objective and a scale bar (50 μm) was depicted on the right bottom corner of the overlay images.
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pone.0140869.g001: The candidate tenogenic markers (COL-I, TNMD, TNC and SCX) expression of GDF5 (100 ng/ml)-induced hMSC on day 4 (A, B, C) and day 10 (D, E, F) by immunofluorescence imaging.The extent of candidate tenogenic markers expressions were increased in GDF5 treated hMSC compared to the untreated control. An increase in the intensity of the expression of these markers was also observed in day 10 GDF5-induced hMSCs compared to that of day 4. Images were captured at 63X objective and a scale bar (50 μm) was depicted on the right bottom corner of the overlay images.

Mentions: Bone marrow derived cells enriched for hMSCs (n = 6) were characterized to confirm their MSC phenotypic markers expression (CD29+, CD44+, CD73+, CD81+, CD90+, CD105+, CD166+, CD14-, CD19-, CD34-, CD45-, CD117- and HLA-DR-) and capability for tri-lineage differentiation (osteogenic, chondrogenic and adipogenic differentiation) as previously described [12]. Tenogenic differentiation was conducted as previously described [2], and verified by immunofluorescence staining for candidate tenogenic markers. The results revealed an increase in candidate tenogenic markers protein expression in hMSCs in day 4 and day 10 GDF5-induced hMSCs compared to control (Fig 1); and considerable similarity in the cellular distribution of candidate tenogenic markers in GDF5-induced hMSCs and tenocytes at day 4 and 10. In addition, the day-10 GDF5-induced hMSCs showed an elongated “tendon cell-like” morphology.


Identification of Pathways Mediating Growth Differentiation Factor5-Induced Tenogenic Differentiation in Human Bone Marrow Stromal Cells.

Tan SL, Ahmad TS, Ng WM, Azlina AA, Azhar MM, Selvaratnam L, Kamarul T - PLoS ONE (2015)

The candidate tenogenic markers (COL-I, TNMD, TNC and SCX) expression of GDF5 (100 ng/ml)-induced hMSC on day 4 (A, B, C) and day 10 (D, E, F) by immunofluorescence imaging.The extent of candidate tenogenic markers expressions were increased in GDF5 treated hMSC compared to the untreated control. An increase in the intensity of the expression of these markers was also observed in day 10 GDF5-induced hMSCs compared to that of day 4. Images were captured at 63X objective and a scale bar (50 μm) was depicted on the right bottom corner of the overlay images.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4631504&req=5

pone.0140869.g001: The candidate tenogenic markers (COL-I, TNMD, TNC and SCX) expression of GDF5 (100 ng/ml)-induced hMSC on day 4 (A, B, C) and day 10 (D, E, F) by immunofluorescence imaging.The extent of candidate tenogenic markers expressions were increased in GDF5 treated hMSC compared to the untreated control. An increase in the intensity of the expression of these markers was also observed in day 10 GDF5-induced hMSCs compared to that of day 4. Images were captured at 63X objective and a scale bar (50 μm) was depicted on the right bottom corner of the overlay images.
Mentions: Bone marrow derived cells enriched for hMSCs (n = 6) were characterized to confirm their MSC phenotypic markers expression (CD29+, CD44+, CD73+, CD81+, CD90+, CD105+, CD166+, CD14-, CD19-, CD34-, CD45-, CD117- and HLA-DR-) and capability for tri-lineage differentiation (osteogenic, chondrogenic and adipogenic differentiation) as previously described [12]. Tenogenic differentiation was conducted as previously described [2], and verified by immunofluorescence staining for candidate tenogenic markers. The results revealed an increase in candidate tenogenic markers protein expression in hMSCs in day 4 and day 10 GDF5-induced hMSCs compared to control (Fig 1); and considerable similarity in the cellular distribution of candidate tenogenic markers in GDF5-induced hMSCs and tenocytes at day 4 and 10. In addition, the day-10 GDF5-induced hMSCs showed an elongated “tendon cell-like” morphology.

Bottom Line: The genes identified as potentially associated with tenogenic differentiation were ARHGAP29, CCL2, integrin alpha 8 and neurofilament medium polypeptides.These genes, were mainly associated with cytoskeleton reorganization (stress fibers formation) signaling.Understanding and elucidating the tenogenic differentiation signalling pathways are important for future optimization of tenogenic hMSCs for functional tendon cell-based therapy and tissue engineering.

View Article: PubMed Central - PubMed

Affiliation: Tissue Engineering Group (TEG), National Orthopaedic Centre of Excellence for Research and Learning (NOCERAL), Department of Orthopaedic Surgery, Faculty of Medicine, University of Malaya, Pantai Valley, Kuala Lumpur, 50603, Malaysia.

ABSTRACT
To date, the molecular signalling mechanisms which regulate growth factors-induced MSCs tenogenic differentiation remain largely unknown. Therefore, a study to determine the global gene expression profile of tenogenic differentiation in human bone marrow stromal cells (hMSCs) using growth differentiation factor 5 (GDF5) was conducted. Microarray analyses were conducted on hMSCs cultures supplemented with 100 ng/ml of GDF5 and compared to undifferentiated hMSCs and adult tenocytes. Results of QuantiGene® Plex assay support the use and interpretation of the inferred gene expression profiles and pathways information. From the 27,216 genes assessed, 873 genes (3.21% of the overall human transcriptome) were significantly altered during the tenogenic differentiation process (corrected p<0.05). The genes identified as potentially associated with tenogenic differentiation were ARHGAP29, CCL2, integrin alpha 8 and neurofilament medium polypeptides. These genes, were mainly associated with cytoskeleton reorganization (stress fibers formation) signaling. Pathway analysis demonstrated the potential molecular pathways involved in tenogenic differentiation were: cytoskeleton reorganization related i.e. keratin filament signaling and activin A signaling; cell adhesion related i.e. chemokine and adhesion signaling; and extracellular matrix related i.e. arachidonic acid production signaling. Further investigation using atomic force microscopy and confocal laser scanning microscopy demonstrated apparent cytoskeleton reorganization in GDF5-induced hMSCs suggesting that cytoskeleton reorganization signaling is an important event involved in tenogenic differentiation. Besides, a reduced nucleostemin expression observed suggested a lower cell proliferation rate in hMSCs undergoing tenogenic differentiation. Understanding and elucidating the tenogenic differentiation signalling pathways are important for future optimization of tenogenic hMSCs for functional tendon cell-based therapy and tissue engineering.

No MeSH data available.


Related in: MedlinePlus