Spontaneous In Vivo Chondrogenesis of Bone Marrow-Derived Mesenchymal Progenitor Cells by Blocking Vascular Endothelial Growth Factor Signaling

Abstract

: Chondrogenic differentiation of bone marrow-derived mesenchymal stromal/stem cells (MSCs) can be induced by presenting morphogenetic factors or soluble signals but typically suffers from limited efficiency, reproducibility across primary batches, and maintenance of phenotypic stability. Considering the avascular and hypoxic milieu of articular cartilage, we hypothesized that sole inhibition of angiogenesis can provide physiological cues to direct in vivo differentiation of uncommitted MSCs to stable cartilage formation. Human MSCs were retrovirally transduced to express a decoy soluble vascular endothelial growth factor (VEGF) receptor-2 (sFlk1), which efficiently sequesters endogenous VEGF in vivo, seeded on collagen sponges and immediately implanted ectopically in nude mice. Although naïve cells formed vascularized fibrous tissue, sFlk1-MSCs abolished vascular ingrowth into engineered constructs, which efficiently and reproducibly developed into hyaline cartilage. The generated cartilage was phenotypically stable and showed no sign of hypertrophic evolution up to 12 weeks. In vitro analyses indicated that spontaneous chondrogenic differentiation by blockade of angiogenesis was related to the generation of a hypoxic environment, in turn activating the transforming growth factor-β pathway. These findings suggest that VEGF blockade is a robust strategy to enhance cartilage repair by endogenous or grafted mesenchymal progenitors. This article outlines the general paradigm of controlling the fate of implanted stem/progenitor cells by engineering their ability to establish specific microenvironmental conditions rather than directly providing individual morphogenic cues.

Significance: Chondrogenic differentiation of mesenchymal stromal/stem cells (MSCs) is typically targeted by morphogen delivery, which is often associated with limited efficiency, stability, and robustness. This article proposes a strategy to engineer MSCs with the capacity to establish specific microenvironmental conditions, supporting their own targeted differentiation program. Sole blockade of angiogenesis mediated by transduction for sFlk-1, without delivery of additional morphogens, is sufficient for inducing MSC chondrogenic differentiation. The findings represent a relevant step forward in the field because the method allowed reducing interdonor variability in MSC differentiation efficiency and, importantly, onset of a stable, nonhypertrophic chondrocyte phenotype.

Keywords: Chondrogenesis; Hypoxia; Mesenchymal stromal/stem cells; Vascular endothelial growth factor blockade.

Figure 1.

Inhibitory effect of sFlk-1 expressed by bone marrow-derived mesenchymal stromal/stem cells (MSCs) on in vitro endothelial organization and proliferation. Human umbilical vein endothelial cell proliferation assay performed by using supernatants collected by naïve (control) or sFlk-1-expressing MSCs. (A): Cell metabolic activity was measured on the basis of the colorimetric MTS assay and represented by using optical density units. (B): Immunofluorescence staining for CD31 in green showing the capillary-like structure formation in the direct coculture of either control or sFlk-1-expressing MSCs with human dermal microvascular endothelial cells. (C): Representative images of tubular structure formation of endothelial cells (stained with calcein assay medium in green after 48 hours) cultured in medium conditioned by control or sFlk-1 expressing MSCs supplemented with 0, 10, or 50 ng/ml of VEGF. Scale bars = 300 µm. (D): Total tube number, length, branching points, and loops were quantified by image analysis. Ten images per sample were analyzed. Data are presented as mean ± SD (n = 4 samples/group from 2 independent experiments). ∗, p < .05; ∗∗, p < .01. Abbreviations: OD, optical density; VEGF, vascular endothelial growth factor.

Figure 2.

In vivo blocking of angiogenesis. (A): Representative macroscopic picture of the explants of engineered tissue generated by naïve (left) or sFlk-1-expressing (right) MSCs after 12 weeks in vivo. (B): Vessel density quantified within the total area of the implant generated by control (naïve) or sFlk-1 MSCs at different time points by histomorphometric analysis (n = 3–4). ∗, p < .05. (C): Immunohistochemistry for mouse CD31. Representative images at ×20 magnification include the central implant area generated by naive (top row) or sFlk-1 (bottom row) MSCs at 1, 4, 8, or 12 weeks. Black arrowheads indicate the blood vessels. Scale bar = 100 µm. Abbreviation: MSC, bone marrow-derived mesenchymal stromal/stem cell.

Figure 3.

In vivo chondrogenesis. Histological staining with Safranin-O for glycosaminoglycans and immunohistochemistry for type II collagen of engineered tissue generated by naïve (control) or sFlk-1 MSCs after 4 (A) or 12 (B) weeks in vivo. Fluorescence staining with DAPI (in blue) and a specific anti-human nuclei antibody (in red) of constructs generated by control or sFlk-1 MSCs after 4 (A) or 12 (B) weeks in vivo. Scale bar = 100 µm. Abbreviations: DAPI, 4′,6-diamidino-2-phenylindole; MSC, bone marrow-derived mesenchymal stromal/stem cell.

Figure 4.

In vivo cartilage stability. Immunohistochemistry for type X collagen, BSP, and MMP-13 on sections of hypertrophic cartilage generated in vitro by MSCs (as a positive control) and on sections of the cartilaginous constructs generated in vivo by sFlk1 MSCs 12 weeks after implantation. Scale bar = 50 µm. Abbreviations: BSP, bone sialoprotein; MMP-13, metalloproteinase-13; MSC, bone marrow-derived mesenchymal stromal/stem cell.

Figure 5.

In vitro chondrogenesis at different oxygen tensions. Histological staining with Safranin-O and immunohistochemistry for type II collagen on constructs generated in vitro by naïve MSC cultured with (A) or without (B) TGFβ3 supplementation at 2% or 20% of oxygen tension. Scale bar = 50 µm. Expression levels of the mRNA for type II and X collagen, Gremlin-1, IHH TGFβ1 were quantified in pellets generated by naïve bone marrow-derived mesenchymal stromal/stem cells (C, D) cultured in the two different oxygen tensions. ∆Ct values were normalized to expression of the GAPDH housekeeping gene, and results are shown as mean ± SD (n = 6 samples/group from 3 independent experiments). ∗, p < .05, ∗∗∗, p < .001. Abbreviations: GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IHH, Indian hedgehog; TGFβ, transforming grown factor-β.