Quantitative Assessment of Cell Signaling Pathways Affecting Stem Cell Differentiation Using Lentiviral Arrays

Loss of gene function is a valuable tool for screening genes in cellular processes such as Mesenchymal Stem Cell (MSC) differentiation. However, the criteria for evaluating gene knockdown are usually based on end-point analysis such as immunostaining and therefore, quantitative, real-time measurement of the process is difficult. To overcome these limitations, we created live cell LentiViral Arrays (LVA) for assessing the effects of certain pathways on MSC differentiation. In particular, we engineered a shRNA encoding dual promoter lentiviral vector (sh-LVDP) that allows real-time monitoring of MSC differentiation and simultaneous gene knockdown thereby, providing quantitative assessment of the effect of various pathways in this process. In this vector, the activity of the aSMA promoter was used as an indicator of myogenic differentiation and constitutive expression of DsRed was used to measure transduction efficiency and to normalize the promoter activity. Finally, different shRNAs were encoded by a doxycycline regulatable H1 promoter within the viral 3' long terminal repeat (LTR).

We found that normalized promoter activity (GFI/RFI) measured by the sh-LVDP vector remained unaffected by lentiviral titer allowing quantitative measurements of MSC differentiation as different genes were knocked down. To demonstrate the utility of this approach we chose to knock down known effectors of the TGF-β1 or Rho signaling pathway (SMAD2, SMAD3, SMAD4, RhoA, Rock1, MRTFB/MLK2, PKN1, SRF, myocardin), which decreased αSMA promoter activity significantly as compared to control (scramble shRNA) (Fig. 1B). On the other hand, knocking down myogenic differentiation inhibitor such as KLF4 increased aSMA promoter activity significantly. Notably, some proteins e.g. SMAD7 or KLF4 showed differential regulation of myogenic differentiation of bone marrow as compared to hair follicle derived MSC. Finally, doxycyline regulatable shRNA expression provided dynamic monitoring of myogenic differentiation as a function of the degree of gene knockdown. Our data indicates that  real time monitoring of  gene expression profiles  using LVA with concomitant gene knockdown provides a useful tool for deciphering gene regulatory networks of complex biological processes such as stem cell differentiation.

 

Figure 1: Lentiviral MicroArray (LVA) with sh-LVDP for differentiation toward smooth muscle cells in response to TGFβ.  (A) Schematic of sh-LVDP vector carrying the aSMA promoter. (B) Normalization of fluorescence intensity (GFI/RFI) indicates the a-actin promoter activity affected by gene knock-down in Human Aortic Smooth Muscle Cells (HASMCs).