Int J Stem Cells.  2019 Mar;12(1):139-150. 10.15283/ijsc18088.

TGF-β Signalling is Suppressed under Pro-Hypertrophic Conditions in MSC Chondrogenesis Due to TGF-β Receptor Downregulation

• Pfeifer CG
• Karl A
• Kerschbaum M
• Berner A
• Lang S
• Schupfner
• Koch M
• Angele P
• Nerlich M
• Mueller MB

• KMID: 2447232
• DOI: http://doi.org/10.15283/ijsc18088

Abstract

BACKGROUND AND OBJECTIVES
Mesenchymal stem cells (MSCs) become hypertrophic in long term despite chondrogenic differentiation following the pathway of growth plate chondrocytes. This terminal differentiation leads to phenotypically unstable cartilage and was mirrored in vitro by addition of hypertrophy inducing medium. We investigated how intrinsic TGF-β signaling is altered in pro-hypertrophic conditions.
METHODS AND RESULTS
Human bone marrow derived MSC were chondrogenically differentiated in 3D culture. At day 14 medium conditions were changed to 1. pro-hypertrophic by addition of T3 and withdrawal of TGF-β and dexamethasone 2. pro-hypertrophic by addition of BMP 4 and withdrawal of TGF-β and dexamethasone and 3. kept in prochondrogenic medium conditions. All groups were treated with and without TGFβ-type-1-receptor inhibitor SB431542 from day 14 on. Aggregates were harvested for histo- and immunohistological analysis at d14 and d28, for gene expression analysis (rt-PCR) on d1, d3, d7, d14, d17, d21 and d28 and for Western blot analysis on d21 and d28. Induction of hypertrophy was achieved in the pro-hypertrophic groups while expression of TGFβ-type-1- and 2-receptor and Sox 9 were significantly downregulated compared to pro-chondrogenic conditions. Western blotting showed reduced phosphorylation of Smad 2 and 3 in hypertrophic samples, reduced TGF-β-1 receptor proteins and reduced SOX 9. Addition of SB431542 did not initiate hypertrophy under pro-chondrogenic conditions, but was capable of enhancing hypertrophy when applied simultaneously with BMP-4.
CONCLUSIONS
Our results suggest that the enhancement of hypertrophy in this model is a result of both activation of pro-hypertrophic BMP signaling and reduction of anti-hypertrophic TGFβ signaling.

Keyword

Mesenchymal stem cells; Chondrogenesis; Terminal differentiation; Receptor

MeSH Terms

Blotting, Western
Bone Marrow
Cartilage
Chondrocytes
Chondrogenesis*
Dexamethasone
Down-Regulation*
Gene Expression
Growth Plate
Humans
Hypertrophy
In Vitro Techniques
Mesenchymal Stromal Cells
Phosphorylation
Dexamethasone

Figure

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  Fig. 1 Differences in glycosaminoglycan content shown by DMMB staining of cell pellets after chondrogenic (A) and hypertrophic conditions (B), as well as different collagen 2 production as revealed by immunohistochemistry against collagen 2 of cell pellets after chondrogenic (C) and hypertrophic conditions (D). Enhancement of hypertrophy shown by immunohistochemistry against collagen 10 between chondrogenic (E) and hypertrophic (F) conditioned cell pellets as well as by ALP staining of chondrogenic (G) and hypertrophic (H) conditioned cell pellets (Scale bar=500 μm).

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  Fig. 2 Gene expression analysis of TGFβR1, TGFβR2 and Sox9 normalized to HPRT in MSC pellet cultures under chondrogenic and hypertrophy enhancing conditions analyzed by real time PCR. TGFβR1 is significantly down-regulated under hypertrophic conditions on day 17, 21 and 28 (A). TGFβR2 is down-regulated under hypertrophic conditions on day 28 (B). Sox9 is down-regulated on day 17 and 21 in hypertrophic MSC pellets (C). n=7 different donors.

• 

  Fig. 3 TGFβ signaling activity. Western Blot analysis of phospho-Smad2 and phospho-Smad3 under chondrogenic and hypertrophic conditions. The amount of phospho-Smad2 and phospho-Smad3 is reduced under hypertrophic conditions on day 21 and day 28 compared to chondrogenic conditions. The total amount of Smad2/Smad3 and β actin were taken as loading control.

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  Fig. 4 Western Blot analysis of TGF βR1 and Sox9. TGFβR1 protein amount is reduced on day 21 and day 28 under hypertrophic conditions (A). Sox9 protein level is decreased on day 21 under hypertrophic conditions (B). β actin was used as loading control.

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  Fig. 5 Histological appearance of MSC pellet cultures on day 28 after SB431542 treatment under chondrogenic conditions. No change in DMMB, ALP, collagen type II and collagen type X staining can be detected between chondrogenic control pellets (A, E, I, M) and pellets treated with low doses of SB431542 (B, C, F, G, J, K, N, O). High doses of SB431542 lead to a dedifferentiation of the cells shown by decreased DMMB staining (D) and collagen type II staining (L) (Scale bar=200 μm).

• 

  Fig. 6 Histological appearance of MSC pellet cultures on day 28 after SB431542 and BMP4 treatment under chondrogenic conditions. Concomitant application of SB431542 and BMP4 increases hypertrophy in a dose dependent manner shown by an increased amount of hypertrophic (B~D) and ALP positive (F~H) cells and increased collagen type X staining (N~P) after SB431542 and BMP4 treatment, compared to BMP4 only treatment (A, E, M). No difference in collagen type II staining can be detected between BMP4 treated control pellets (I) and pellets treated with SB431542 and BMP4 (J~L) (Scale bar=200 μm).

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  Fig. 7 Histological appearance of MSC pellet cultures on day 28 after SB431542 treatment under hypertrophic conditions. No difference in DMMB, ALP, collagen type II and collagen type X staining can be detected between hypertrophic control pellets (A, E, I, M) and SB431542 treated pellets (B~D, F~H, J~L, N~P) (Scale bar=200 μm).

Reference

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