shRNA-mediated decreases in c-Met levels affect the differentiation potential of human mesenchymal stem cells and reduce their capacity for tissue repair

Abstract

Mesenchymal stem cells/marrow stromal cells (MSC) are adult multipotent cells that can augment tissue repair. We previously demonstrated that culturing MSC in hypoxic conditions causes upregulation of the hepatocyte growth factor (HGF) receptor c-Met, allowing them to respond more robustly to HGF. MSC preconditioned in hypoxic environments contributed to restoration of blood flow after an ischemic injury more rapidly than MSC cultured in normoxic conditions. We now investigated the specific role of HGF/c-Met signaling in MSC function. An shRNA-mediated knockdown (KD) of c-Met in MSC did not alter their phenotypic profile, proliferation, or viability in vitro. However, we determined that while HGF/c-Met signaling does not play a role in the adipogenic differentiation of the cells, the disruption of this signaling pathway inhibited the ability of MSC to differentiate into the osteogenic and chondrogenic lineages. We next assessed the impact of c-Met KD on human MSC function in a xenogeneic hindlimb ischemia injury model. A 70% KD of c-Met in MSC resulted in a significant decrease in their capacity to regenerate blood flow to the ischemic limb, as compared to the MSC transduced with control shRNA. MSC with only a 60% KD of c-Met exhibited an intermediate capacity to restore blood flow, suggesting that MSC function is sensitive to the dosage of c-Met signaling. The current study highlights the significance of HGF/c-Met signaling in the capacity of MSC to restore blood flow after an ischemic injury and in their ability to differentiate into the osteogenic and chondrogenic lineages.

FIG. 1.

Western blot analysis of shRNA-mediated c-Met KD. (a) MSC were transduced with lentivirus expressing shRNA constructs targeting HGF-receptor c-Met or scrambled control. The level of c-Met KD was assessed after the puromycin selection using Western blot analysis. Both vectors 4246 and 4571 resulted in observable KD, whereas the scrambled control did not affect the c-Met protein levels on MSC. (b) The KD of c-Met was verified after every transduction, and the level of KD was semi-quantitated using densitometry analysis (Image J). C-Met semi-quantification was normalized by tubulin expression. There was no significant difference in the level of reduction in protein levels between the two vectors (p > 0.05), while both were significantly decreased as compared to the scrambled control (p < 0.05). The data are representative of nine transductions of primary MSC with the scrambled, 4246, and 4571 vectors. MSC, mesenchymal stem cells/marrow stromal cells; HGF, hepatocyte growth factor; KD, knockdown; BMSC, bone-marrow-derived MSC.

FIG. 2.

Marker profile of KD MSC. The cell surface marker profile of KD versus scrambled shRNA MSC (KD MSC and SCR MSC, respectively) was analyzed using flow cytometry analysis. There was no difference between KD MSC and SCR MSC in their cell surface marker expression. Consistent with MSC characteristics, MSC lacked hematopoietic markers CD34 and CD45 (a) and expressed MSC markers CD105 and CD90 (b) and CD73 (c). The gates were set based on the analysis of cells stained with IgG controls, as shown in (d), (e), and (f), respectively. The images are representative of three separate experiments. The summary of the flow cytometry data (% of cells expressing the marker ± standard deviation) is shown in Table 1. SCR, scrambled control shRNA.

FIG. 3.

c-Met KD does not alter MSC cell cycle, survival, or colony-forming efficiency. (a) Cell cycle: KD MSC or SCR MSC were permeabilized, stained with propidium iodide and their cell cycle status was assessed based on their DNA content. The bar graph summarizes the results of three separate experiments, showing that there was no significant difference in proliferation between KD MSC and SCR MSC. (b) Survival: KD or SCR MSC were collected, stained with Annexin V and 7AAD, and analyzed by flow cytometry. The bar graph summarizes the proportion of dead and apoptotic cells from three separate experiments, and demonstrates no significant differences. (c) CFU-F assay: MSC were plated at low density (500 cells/well of six-well plate) and the colonies were counted after 14 days of culture. The bar graph summarizes the data from two separate experiments (n = 6 for each treatment), showing that although there was a significant difference between transduced and untransduced MSC (*), there was no significant difference between KD MSC and SCR MSC as assessed using one-way ANOVA. CFU-F, colony forming unit-fibroblast; ANOVA, analysis of variance.

FIG. 4.

HGF/c-Met signaling alters the osteogenic and chondrogenic differentiation capacity of MSC, but not their adipogenic differentiation potential. MSC were cultured in the commercial differentiation medium to stimulate differentiation along adipogenic, osteogenic, and chondrogenic lineages. (a) Adipogenic cultures were stained with Oil Red O, showing no differences between c-Met KD MSC and SCR MSC in their adipogenic differentiation capacity. (b) Alizarin Red S staining of calcium deposits revealed a decrease of osteogenic differentiation in KD MSC compared to SCR MSC and untransduced MSC. (c) Safranin O staining of chondrogenic pellets demonstrated a decrease in chondrogenic differentiation potential in KD MSC compared to SCR MSC and untransduced MSC. Notably, the level of differentiation of SCR MSC in all three differentiation assays was comparable to the untransduced MSC. Shown are representative images of 5–7 replicates of osteogenic and adipogenic cultures performed in three separate experiments. The chondrogenic assays were performed as three independent replicates.

FIG. 5.

The effect of HGF/c-Met signaling on MSC tissue repair capacity in a hindlimb ischemia injury model. (a) Hindlimb ischemia surgeries were performed on immunodeficient NOD/SCID mice, followed by intraperitoneal injection of anti-CD122 antibody. Approximately 24 h after the surgery, the mice were injected intramuscularly with 10⁶ human BMSC that had been cultured at 3% oxygen or 21% oxygen. The blood flow recovery to the ischemic limb was followed by laser Doppler perfusion imaging for 2 weeks after the surgery. (b) The laser Doppler perfusion imaging results demonstrate that the mice injected with hypoxia-preconditioned MSC recovered significantly better than the saline controls (two-way ANOVA), whereas there was no significant difference between the control group and the group injected with MSC cultured in 21% oxygen (two-way ANOVA). On the basis of these results, the MSC used in our next hindlimb ischemia experiment were all preconditioned at 1%–3% oxygen before the transplantation. (c) Mice injected with SCR MSC were able to improve blood flow recovery after hindlimb ischemia to the same degree as nontransduced MSC; both SCR- and nontransduced MSC restored blood flow significantly faster than the saline controls (two-way ANOVA). The blood flow recovery in mice transplanted with 4246 KD MSC (70% KD of c-Met) was significantly slower than that in the SCR MSC group (two-way ANOVA). (d) The blood flow recovery of animals treated with 4571 KD MSC (60% c-Met KD) was not significantly different from those treated with SCR MSC. The summarized data are based on seven (b, hypoxic and PBS groups) and eight (b, normoxic) animals per group. The graphs shown in (c) and (d) belong to the same experiment and are only separated to improve clarity. Therefore, these data can be directly compared to each other, and the SCR MSC and PBS curves are identical in both graphs. The data are representative of 12 (4246), 14 (untransduced MSC and SCR MSC), and 15 (PBS and 4571) animals per group. *Signifies p < 0.05 compared with saline-treated mice. NS, not significant. PBS, phosphate-buffered saline. Color images available online at www.liebertonline.com/ten.