Transfection of MSCs with RNAis for PODXL. Viable MSCs from passage 1/donor 1 were plated at 100 cells/cm2, incubated for 5 days, transfected for 5 hours, and then incubated in CCM for 2 days. (A) mRNA for PODXL assayed by real-time RT-PCR from normal control MSCs and transfected MSCs with single PODXL RNAi, a mixture of 3 PODXL RNAis or a RNAi negative control. Values are mean plus or minus SD (n = 3). (B) FACScan for PODXL of MSCs incubated with 20 nmol/L RNAis for PODXL as in panel A. (C) Phase-contrast microscopy of control MSCs and MSCs transfected with 20 nmol/L of mixture of 3 RNAis for PODXL and then incubated for 2 days. Bar = 100 μm. (D) Real-time RT-PCR assays for PODXL mRNA of cultures that were transduced with mixture of 3 RNAis and incubated as in panel C, lifted with trypsin/EDTA, replated at approximately 1000 cells/cm2, and incubated for 24 hours. Cells from regions of the plate containing single cells and aggregated cells were isolated separately by using cloning cylinders and assayed separately. Values are mean plus or minus SD (n = 3). (E) Phase microscopy of cultures taken for RT-PCR assays in panel D. Region containing single cells and 2 aggregates are shown. Bar = 100 μm. (F) Immunocytochemistry for control culture and transduced cultures shown in panel E. The dispersed single cells (middle panel) were lightly labeled with anti-PODXL. In the aggregates only the peripheral cells were labeled (right panel). Nuclei were stained with DAPI. Bar = 100 μm.
