MMP-2, MT1-MMP, and TIMP-2 are essential for the invasive capacity of human mesenchymal stem cells: differential regulation by inflammatory cytokines by Christian Ries, Virginia Egea, Marisa Karow, Helmut Kolb, Marianne Jochum, and Peter Neth Blood Volume 109(9):4055-4063 May 1, 2007 ©2007 by American Society of Hematology

Constitutive expression of mRNA and protein of MMPs and TIMPs in hMSCs. Constitutive expression of mRNA and protein of MMPs and TIMPs in hMSCs. (A) qRT-PCR analysis of MMP-2, MMP-9, MT1-MMP, TIMP-1, and TIMP-2 gene transcription in hMSCs cultivated in MSCG medium. The results are mean values ± SDs of mRNA expression relative to GAPDH (set as 1) from a triplicate measurement representative for 3 independent experiments with 3 different hMSC lots. (B) Western blot detection of MMP-2, MMP-9, MT1-MMP, TIMP-1, and TIMP-2 in cell lysates (LY) and conditioned medium (CM) of hMSCs cultivated for 72 hours under serum-free conditions. Aliquots (30 μL) standardized by protein content were separated by SDS-PAGE under reducing conditions, blotted, and probed with the specific antibodies. (C) Zymographic analysis of gelatinase secretion from hMSCs. Aliquots of culture supernatants (10 μL) taken at different time points during a 72-hour cultivation period of hMSCs in serum-free medium were analyzed. HT1080 conditioned medium containing proMMP-9, proMMP-2, and active forms of MMP-2 was used as a marker (M).37 Christian Ries et al. Blood 2007;109:4055-4063 ©2007 by American Society of Hematology

Influence of MMP inhibitors on hMSC invasion through a barrier of reconstituted human basement membrane. hMSCs were placed onto Transwell filters coated with human ECM and incubated for 48 hours in the absence (control) or presence of the broad-spectrum MMP... Influence of MMP inhibitors on hMSC invasion through a barrier of reconstituted human basement membrane. hMSCs were placed onto Transwell filters coated with human ECM and incubated for 48 hours in the absence (control) or presence of the broad-spectrum MMP inhibitor GM6001 (10 μg/mL) or Ro 206-0222 (10 μg/mL), a highly specific inhibitor of MMP-2, MMP-9, and MT1-MMP.41 Cell invasion rate was determined in the percentage relative to control (set as 100%). Data are presented as mean ± SD of 1 triplicate experiment representative of 3 independent experiments. **P < .01. Christian Ries et al. Blood 2007;109:4055-4063 ©2007 by American Society of Hematology

MMP/TIMP knock-downs in hMSCs: effect on mRNA and protein levels. MMP/TIMP knock-downs in hMSCs: effect on mRNA and protein levels. (A) hMSCs were transfected with siRNAs targeting the gene expression of MMP-2 (i), MT1-MMP (ii), TIMP-1 (iii), or TIMP-2 (iv). Control cells were transfected with non–target-directed siRNA (set as 100%). Transcription of specific mRNAs was quantified by qRT-PCR 24 and 72 hours after siRNA transfection. Data represent the mean ± SD of a triplicate measurement representative for 5 transfection experiments. ***P < .001, **P < .01. (B) hMSCs transfected with non–target-directed control siRNA (NC) or with siRNA against MMP-2 (KD) were cultivated under serum-free conditions and analyzed for secreted proMMP-2 after different time intervals by zymography. For densitometric quantification, enzyme release from control cells transfected with non–target-directed siRNA was set as 100% at each time point. (C) Western blotting analysis of protein extracts obtained from hMSCs 72 hours after transfection with control siRNA (NC) or with siRNA against MT1-MMP (KD). Cellular actin was detected on the same blot to control for application of equal amounts of protein in each lane. TIMP-1 and TIMP-2 secretion from hMSCs carrying the respective knock-downs (KD) or control siRNA (NC) was examined by Western blotting of 72-hour culture supernatants. Protein data are representative of 3 independent experiments with similar results. Christian Ries et al. Blood 2007;109:4055-4063 ©2007 by American Society of Hematology

Effect of MMP/TIMP knock-downs on hMSC invasion Effect of MMP/TIMP knock-downs on hMSC invasion. hMSCs carrying knock-downs for the expression of MMP-2, MT1-MMP (MT1), TIMP-1, and TIMP-2 were assessed for their ability to traverse human ECM. The hMSCs were applied in the Transwell invasion assay 24 hours... Effect of MMP/TIMP knock-downs on hMSC invasion. hMSCs carrying knock-downs for the expression of MMP-2, MT1-MMP (MT1), TIMP-1, and TIMP-2 were assessed for their ability to traverse human ECM. The hMSCs were applied in the Transwell invasion assay 24 hours after their transfection and incubated for 48 hours. Thereafter, cells that had migrated to the lower chamber were counted. Control cells transfected with non–target-directed siRNA were set as 100%. Data are presented as mean ± SD of 1 triplicate experiment representative of 3 independent measurements. ***P < .001, **P < .01, *P < .05. Christian Ries et al. Blood 2007;109:4055-4063 ©2007 by American Society of Hematology

Influence of cytokines/chemokine on mRNA and protein expression of MMPs/TIMPs in hMSCs. hMSCs were incubated with TGF-β1 (100 ng/mL), IL-1β (50 ng/mL), TNF-α (50 ng/mL), SDF-1α (100 ng/mL), or left untreated (control, Con) and cultivated under serum-free co... Influence of cytokines/chemokine on mRNA and protein expression of MMPs/TIMPs in hMSCs. hMSCs were incubated with TGF-β1 (100 ng/mL), IL-1β (50 ng/mL), TNF-α (50 ng/mL), SDF-1α (100 ng/mL), or left untreated (control, Con) and cultivated under serum-free conditions for up to 72 hours. (A) mRNA expression of MMP-2, MMP-9, MT1-MMP (MT1), TIMP-1, and TIMP-2 were quantified by qRT-PCR after 24 and 72 hours of incubation with cytokines/chemokine. Results are given as the percentage of change in mRNA expression relative to untreated cells set as 100%. Values represent the mean ± SD of 1 triplicate experiment from 2 independent measurements. (B-C) Aliquots of 72-hour culture supernatants were subjected to zymography. Medium samples were applied in a 1:4 dilution to allow densitometric quantification of proMMP-2 (B) or undiluted to achieve higher sensitivity in the detection of (pro)MMP-9 (C). (D) MT1-MMP protein was determined in cell lysates of hMSCs incubated for 72 hours with and without cytokines/chemokine using the MMP-14 Biotrak Activity Assay. Data are shown as mean ± SD of 1 of 2 triplicate experiments performed. ***P < .001. Christian Ries et al. Blood 2007;109:4055-4063 ©2007 by American Society of Hematology

Chemotactic importance of cytokines/chemokine on hMSC invasion and the implication of MMPs. hMSCs were seeded in the upper compartment of Transwell cell invasion chambers. Chemotactic importance of cytokines/chemokine on hMSC invasion and the implication of MMPs. hMSCs were seeded in the upper compartment of Transwell cell invasion chambers. The lower compartment was filled with DMEM containing TGF-β (100 ng/mL), IL-1α (50 ng/mL), TNF-α (50 ng/mL), or SDF-1α (100 ng/mL) as chemoattractants. Control wells contained DMEM medium only (Con). Both upper and lower compartments were provided without (▪) and with Ro 206-0222 (10 μg/mL) to inhibit MMP-2, MMP-9, and MT1-MMP activity (⊡). After a 48-hour period of incubation the amount of migrated cells was quantified. Results are given in fold increase relative to spontaneous cell migration in control wells. All experiments were performed in triplicate. The mean values ± SDs of 1 of 2 separate experiments are shown. *P < .05, **P < .01, ***P < .001. Christian Ries et al. Blood 2007;109:4055-4063 ©2007 by American Society of Hematology