Table 1.
List of Promoters and Response Elements cloned into LVDP.
Fig 1.
Normalized Promoter Activity is independent of gene copies per cell.
(A) Schematic of LVDP-pACTA2-ZsGreen. CMV–promoter sequence from cytomegalovirus; sMAR8 –synthetic MAR sequence 8; TKpA–thymidine kinase polyA from herpes simplex virus; DRE2 –DsRed Express 2; hPGK–human phosphoglycerate kinase promoter; cHS4 –chicken hypersensitive site 4; Tactb−a G-rich sequence from the extension of β-actin gene; SPA–synthetic poly A; ZsG–ZsGreen; pACTA2 –α smooth muscle actin promoter; 5’ LTR: CMV + R + U5; 3’LTR: U3 + R +U5 (B-D) hBM-MSCs were transduced with LVDP-pACTA2-ZsGreen at two different concentrations that differed by 10-fold (1X and 10X). Both red and green fluorescence intensity were measured upon induction to myogenic differentiation. (B) Red Fluorescence Intensity (RFI), (C) Green Fluorescence Intensity (GFI), and (D) Normalized Intensity were plotted against time. Normalized intensity was obtained by dividing GFI by a weighted average of RFI over time, where the weights were determined from a beta distribution (see Materials and Methods) such that the median absolute standard deviation was minimized from the LOESS fit. Data shown are representative of at least three experiments performed with similar results. Ϯ p-value > 0.05.
Fig 2.
Optimal conditions for myogenic differentiation.
Optimized myogenic differentiation medium was determined by treating LVDP-pACTA2-ZsGreen transduced hBM-MSCs with (A) varying concentrations of TGF-β1 (0, 1, 2, 5, 10, and 20 ng/ml) or (B) a combination of 10 ng/ml TGF-β1 (T) and one of the following soluble factors: 30 μg/ml Heparin (H), 30 μM Ascorbic Acid (AA), or 2 μM Insulin (I). Red and green fluorescence intensities were measured by flow cytometry after 2 days and normalized intensity was shown. (A) * denotes p < 0.05 as compared to 10 ng/ml TGF-β1, Ϯ denotes p > 0.05 as compared to 10 ng/ml TGF-β1. (B) * denotes p < 0.05 as compared to T+H. (C) Immunostaining for SMC proteins (αSMA, smooth muscle myosin heavy chain (SM-MHC) and Calponin) under GM or the optimized differentiation medium (DM, T+H) for 7 days. Cell nuclei were counterstained with the Hoechst 33342. Images are representative of three independent experiments. Scale bar: 40 μm.
Fig 3.
High throughput monitoring of Pr/RE activity.
(A) Schematics of Lentiviral Array. (A) Pr/RE of genes potentially involved in myogenic differentiation were cloned in the LVDP. Recombinant lentiviruses were generated and used to transduce MSCs in 384 well plates. Upon successful transduction as evidenced by red fluorescence expression, MSCs were induced to myogenic differentiation. Red and green fluorescence were imaged every 8 hr using fluorescence microscope with automated stage for 6–7 days. GFI and RFI for each image were quantified by CellProfiler. (B) Representative red (row 1 and 3) and green (row 2 and 4) fluorescence images of hBM-MSCs transduced with LVDP-pACTA2-ZsGreen under growth (GM, row 1 and 2) or differentiation (DM, row 3 and 4) conditions at the indicated time (0, 2, 4 and 6 days) of treatment with DM. Scale bar: 500 μm.
Fig 4.
Standardized fold change (Welch’s t-statistic) of normalized intensity in DM over GM for (A) hBM-MSC or (B) hHF-MSC was plotted as a function of time for 27 Pr/RE. The color bar represents the standardized fold change on a scale of (A) -3 to +20 and (B) -5 to +16. The color gradient ranges from blue (no significant change) to red (highly up-regulated).
Fig 5.
Dynamic response of Pr/RE in MSCs during Myogenic Differentiation.
(A, C, E, G, I, K, M, O) hBM-MSCs or (B, D, F, H, J, L, N, P) hHF-MSCs were transduced with the LV library of 27 Pr/RE and coaxed to differentiate into SMCs. The activities of the (A, B) SMAD2/3-RE; (C, D) SMAD7-RE; (E, F) CArG-RE; (G, H) CArGA-RE; (I, J) KLF4-RE; (K, L) ACTA2-Pr; (M, N) SM22-Pr; and (O, P) rMYH11-Pr were determined by fluorescence microscopy and the normalized intensity was plotted as a function of time. MSCs cultured in GM served as a control. * indicates p < 0.05 between DM and GM as determined by Student’s two-tailed t-test at individual time points. + indicates statistical significance of the Pr/RE activities under DM vs GM evaluated over entire curve by growth curve analysis (p < 0.05).
Fig 6.
Effects of signaling pathways on Pr/RE activity.
Dynamics of Pr/RE activity in hHF-MSCs cultured in DM containing chemical inhibitors. (A, B) 10 μM SB431542 (SB4); (C, D) 10 μM CCG1423 (CCG); (E, F) 10 μM Y27632 (Y27); (G, H) 20 μM SB203580 (SB2); (I, J) 10 μM SP600125 (SP6). MSCs cultured in GM served as negative control and MSCs cultured in DM served as positive control. Normalized activities of (A, C, E, G, I) CArG-RE, and (B, D, F, H, J) ACTA2-Pr are shown. The normalized values were scaled from 0–1 and plotted as a function of time. * indicates p < 0.05 between DM and GM as determined by Student’s two-tailed t-test at individual time points. + indicates statistical significance of the Pr/RE activities under DM vs GM evaluated over the entire curve by growth curve analysis (p < 0.05).
Fig 7.
Effects of pathway inhibition on CArG-RE and ACTA2-Pr activity and αSMA protein levels.
Response of (A) CArG-RE and (B) ACTA2-Pr on day 7 of differentiation in the presence of the indicated inhibitors. (C) Western blot for αSMA on day 7 of differentiation in the presence of the indicated inhibitors. GAPDH served as loading control. (D) Relative expression of αSMA as determined from the band intensities in (C). Normalized intensities were scaled to the intensity in DM without inhibitors. * denotes statistical significance (p<0.05, n = 3) as compared to DM.
Fig 8.
Signaling pathways during myogenic differentiation of MSCs.
Signaling pathways during SMC differentiation were compiled from literature [2, 31, 32, 47–50]. External factors (TGFβ, S1P etc) bind to their respective cell surface receptors and activate the downstream signaling pathways that translocate the transcription factors into the nucleus, which then activate the SMC markers. Solid arrows indicate the signaling to the downstream targets in the cytoplasm. Dotted arrows indicate nuclear translocation. Red lines with solid circle end represent inhibition of activity of the target molecule. Normalized fold changes (NIDM/GM) for Pr/RE in hBM-MSCs were mapped onto the pathway. Red and blue font indicates significant up-regulation in hBM-MSC and hHF-MSC, respectively while red font indicates no significant response in DM in hBM-MSC and hHF-MSC, respectively. AII–Angiotensin II; S1P-Sphingosine-1-phosphate; BMP–Bone Morphogenetic Protein; BMPRII–Bone morphogenetic protein receptor II; TGFβ–Transforming Growth Factor β; TGFβR II–Type II Transforming Growth Factor β Receptor; FGF–Fibroblast Growth Factor; P–Phosphorylation. Gene names and descriptions are provided in S6 Table.