(89e) 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.