(210b) High Throughput Monitoring of Pathway Activation Upon Ectopic Expression of Nanog in Human Mesenchymal Stem Cells Using Lentiviral Arrays

Nanog
has been shown to regulate the proliferation capacity of Mesenchymal Stem Cells
(MSCs) and alter their differentiation potential as assessed by overexpression
and knock-down studies. Overexpression of Nanog in MSCs is known to reduce their
adipogenic differentiation potential while inducing osteogenesis. Our laboratory
has shown that Nanog overexpression in MSCs enhances their myogenic
differentiation potential as evidenced by improved contractility. However the
molecular mechanisms by which Nanog plays a specific role in differentiation are
not known. In this study we present a novel tool for high throughput screening
of different pathways in Nanog expressing MSCs. We employed a novel
dual-promoter lentiviral vector (LVDP) carrying human PGK promoter driving the
expression of DsRed serving as an internal control and the promoter of interest
driving the expression of ZsGreen to monitor gene expression upon induction of
Nanog expression in human Hair Follicle derived MSCs (hHF-MSCs) and Bone Marrow
derived MSCs (hBM-MSCs). We employed a Tet-On system for induced expression of
Nanog in hHF-MSCs and hBM-MSCs in the presence of doxycycline. The MSCs were transduced
with a lentiviral library of about 60 vectors containing promoters and
transcriptional response elements (RE) targeting about 25 different pathways
and lineage specific markers. The cells were subsequently induced to myogenic
differentiation and the promoter/RE activity
was monitored over time with automated fluorescence microscope. Fluorescence
intensity was quantified by Cell Profiler and the green fluorescence intensity
was normalized by red fluorescence intensity. First, we used the lentiviral library to monitor gene expression
dynamics during myogenic differentiation of hHF-MSCs and hBM-MSCs over a period
of 7 days. Smooth muscle cell markers such as α-SMA, Myocardin, RE-CArG, and
components of TGF-β1 signaling pathway such as RE-SMAD2/3 and RE-SMAD7 showed
significant upregulation. In the presence of TGF-β1 receptor inhibitor
SB431542, RE-SMAD2/3 and RE-SMAD7 did not respond, indicating that the pathway
was shutdown as expected. We then employed the lentiviral array to identify which
pathways were activated upon ectopic expression of Nanog. Overexpression of
Nanog alone induced a marked response in RE-CArG within 3 days showing an
enhanced potential for myogenic differentiation (Figure 1). Treatment of
Nanog⁺ cells with TGF-β1 and Heparin further increased the
activity of RE-CArG, suggesting that effects of Nanog and TGF-β1 in
promoting MSC myogenesis were additive. RE-ERK representing the MAPK signaling
pathway showed an increased activity in Nanog⁺ cells. However treatment
of Nanog⁺ cells with TGF-β1 blocked its response. RE-STAT3 (JAK/STAT
signaling pathway), RE-KLF4, RE-GATA and RE-GATA2 also showed significant
activation in Nanog⁺ cells but treatment with TGF-β1 did not increase
their response any further. We thus demonstrate the application of lentiviral
arrays in monitoring gene expression during myogenic differentiation of
hHF-MSCs and hBM-MSCs. We present a novel screening strategy to identify
various pathways that are activated upon ectopic expression of Nanog in MSCs.
Currently we are applying this approach to study the role
of Nanog in osteogenic, chondrogenic and adipogenic differentiation.