CTGF directs fibroblast differentiation from human mesenchymal stem/stromal cells and defines connective tissue healing in a rodent injury model

Abstract

Fibroblasts are ubiquitous cells that demonstrate remarkable diversity. However, their origin and pathways of differentiation remain poorly defined. Here, we show that connective tissue growth factor (CTGF; also known as CCN2) is sufficient to induce human bone marrow mesenchymal stem/stromal cells (MSCs) to differentiate into fibroblasts. CTGF-stimulated MSCs lost their surface mesenchymal epitopes, expressed broad fibroblastic hallmarks, and increasingly synthesized collagen type I and tenacin-C. After fibroblastic commitment, the ability of MSCs to differentiate into nonfibroblastic lineages - including osteoblasts, chondrocytes, and adipocytes - was diminished. To address inherent heterogeneity in MSC culture, we established 18 single MSC-derived clones by limiting dilution. CTGF-treated MSCs were alpha-SMA-, differentiating into alpha-SMA+ myofibroblasts only when stimulated subsequently with TGF-beta1, suggestive of stepwise processes of fibroblast commitment, fibrogenesis, and pathological fibrosis. In rats, in vivo microencapsulated delivery of CTGF prompted postnatal connective tissue to undergo fibrogenesis rather than ectopic mineralization. The knowledge that fibroblasts have a mesenchymal origin may enrich our understanding of organ fibrosis, cancer stroma, ectopic mineralization, scarring, and regeneration.

Figure 1. CTGF-mediated fibroblastic differentiation of MSCs.

(A) Bone marrow MSCs were isolated and culture expanded. (B) CTGF treatment (100 ng/ml) prompted substantial collagen synthesis (Masson trichrome) compared with MSCs without CTGF treatment (A). (C and D) Col-I (C) and Tn-C (D) contents of CTGF-treated MSCs cells were significantly higher than MSCs without CTGF treatment at the 2- and 4-week time points (n = 5), as determined by ELISA. (E) Collagen deposition increased with increasing CTGF doses from 0 to 100 ng/ml (Goldner trichrome). (F) Levels of MSC surface epitopes were gradually attenuated, including CD29, CD44, CD105, CD106, CD117, BMPR1A, and Sca1 upon 2 and 4 weeks of CTGF treatment. (G) Concurrently, levels of fibroblastic markers gradually increased, including Col-I, Col-III, Tn-C, fibronectin (FN), MMP-1, FSP1, and vimentin (VIM) upon 4 weeks of CTGF treatment. The chondrogenic marker Col-II and myofibroblastic marker α-SMA were undetectable, whereas the osteogenic marker osteocalcin (OC) was minimally expressed (G). Scale bars: 100 μm. Data represent mean ± SD. *P < 0.05.

Figure 2. CTGF-derived fibroblasts are a stable population.

(A–F) MSC-derived fibroblasts (MSC-Fb) by CTGF treatment for 4 weeks showed minimal capacity to further differentiate into osteoblasts (A), chondrocytes (B), or adipocytes (C). In contrast, native MSCs, without CTGF treatment, readily differentiated into osteoblasts (D), chondrocytes (E), and adipocytes (F). (G–I) von Kossa staining was negative in CTGF-treated MSCs (H), just as MSCs without CTGF treatment (G). (I) In contrast, MSCs subjected to osteogenic stimulation readily differentiated into osteogenic cells that elaborated minerals. (J–L) Safranin O staining was negative in CTGF-treated MSCs (K), just as in MSCs without CTGF treatment (J). (L) In contrast, MSCs subjected to chondrogenic stimulation readily differentiated into chondrogenic cells that were safranin O positive. (M) Quantitatively, MSCs under osteogenic stimulation (MSC-Ob) elaborated significantly more calcium than did MSCs with or without CTGF treatment (n = 5). (N) In parallel, MSCs under chondrogenic stimulation (MSC-Ch) produced significantly more glycosaminoglycans (GAG) than MSCs with or without CTGF treatment (n = 5). Scale bars: 100 μm (A, B, D, E, and G–L); 50 μm (C and F). Data represent mean ± SD. *P < 0.05; **P < 0.01.

Figure 3. Clonal progenies of MSCs differentiate into multiple mesenchymal lineages.

To address the heterogeneity of typical culture of MSCs by adherence to cell culture polystyrene, we established a total of 18 single cell–derived clones from 43 plated wells (approximately 42%) by limiting dilution. Of the 18 isolated clones, 12 clonal progenies (about 67%) differentiated into all of fibroblastic, osteogenic, chondrogenic, and adipogenic cells, whereas a total of 2 clonal progenies (about 11%) differentiated into fibroblastic, osteogenic, and chondrogenic cells, but not into adipogenic cells. The remaining 4 clonal progenies (about 22%) failed to differentiate into any of fibroblastic, osteogenic, chondrogenic, or adipogenic cells. (A–L) Clones, B7, B12, and E3 are shown. All 3 tested clones readily differentiated into fibroblast-like cells that elaborated collagen (A–C); osteogenic cells that produced alkaline phosphatase and minerals (D–F); adipogenic cells that were Oil Red O positive (G and H), with a notable exception of E3 (I); and chondrogenic cells that were safranin O positive (J–L). Scale bars: 100 μm (A–F and J–L); 50 μm (G–I).

Figure 4. Myofibroblastic differentiation of MSC-derived fibroblastic cells by TGF-β1.

(A–D) Native MSCs (A) or MSC-derived fibroblasts by CTGF treatment (C) expressed little α-SMA. Upon TGF-β1 treatment, native MSCs still expressed little α-SMA (B), but MSC-derived fibroblasts readily expressed α-SMA⁺ microfilaments (D). (E–H) Flow cytometry confirmed the virtual absence of α-SMA expression in MSCs (E) or MSC-derived fibroblasts (G). In contrast, 31.9% of MSC-derived fibroblasts (H), but only 1.8% of native MSCs (F), gained α-SMA phenotype after TGF-β1 stimulation. (I–L) Collagen gel contraction assay showed that MSCs with sequential administration of CTGF (4 weeks) and TGF-β1 (1 week) yielded the most significant contraction (I), compared with moderate contraction upon CTGF stimulation alone (J) or TGF-β1 stimulation alone of native MSCs (K). MSCs without either CTGF or TGF-β1 stimulation yielded the least contraction (L). (M) Quantitatively, sequential stimulation of MSCs by CTGF and TGF-β1 yielded the most significant collagen gel contraction (P < 0.05). Scale bars: 100 μm. Data represent mean ± SD.

Figure 5. CTGF induced fibrogenesis instead of ectopic mineralization in vivo.

(A and B) Representative 3D-reconstructed μCT images after resection of a synostosed calvarial suture. Without CTGF, ectopic mineralization (boxed region) was readily observed (A), whereas anatomic morphology was restored in the absence of ectopic mineralization upon controlled release of CTGF (B). (C and D) CTGF-encapsulated PLGA microspheres were 120 ± 64 μm in diameter per scanning EM (C) and showed sustained release up to 6 weeks in vitro (D) (n = 6). (E–H) H&E staining showed that microscopic morphology of the calvarial suture was restored upon controlled release of CTGF (F and H), compared with ectopic mineralization without CTGF delivery (E and G). Some CTGF-encapsulated microspheres (μs) remained present at 4 postoperative weeks (F and H). (I–L) Abundant expression of FSP1 (J) and vimentin (L) indicated the presence of fibroblast-like cells in regenerated calvarial suture; without CTGF delivery (I and K), expression was restricted to the marrow (m) of obliterated bone (b). Scale bars: 1 mm (A and B); 500 μm (C, E, and F); 200 μm (G–L). Data represent mean ± SD.