(425c) Live-Cell Screens for Studying Regulatory Networks in Human Mesenchymal Stem Cell Differentiation

Mesenchymal stem cells (MSCs) are
abundant and multipotent cells with great potential for regenerative medicine
and tissue engineering. The conventional tools for investigating gene
expression such as qPCR are laborious, cost intensive and they cannot provide
functional information on the pathways involved in biological processes. On the
other hand, LentiViral microArrays (LVA) provide
live-cell, high-throughput monitoring of activation of biological pathways
during biological processes such as MSC differentiation. In this paper, we
employed LVA with a library of lentiviral vectors to monitor pathway activation
during MSC differentiation into bone, fat or cartilage. In addition, we
hypothesized that use of LVA in combination with a small molecular chemical library
might identify pathways that control MSC fate commitment.

To address this hypothesis, first
we engineered a dual-promoter lentiviral vector?LVDP containing a specific
transcriptional regulatory element encoding for green fluorescence protein for
monitoring gene regulation. Additionally this vector contains a constitutive
promoter driving red fluorescence protein providing an internal normalization
strategy, thereby allowing measurements of gene or pathway dynamics independent
of the gene transfer efficiency. We developed a library of lineage specific reporters
containing promoters such as adiponectin,
SOX9 and Runx2 for adipogenesis, chondrogenesis and osteogenesis, respectively
as well as transcription factor response motifs e.g. Vitamin D, SMAD, C/EBP
etc. By employing automated fluorescence microscopy, we determined the kinetics
of lineage-specific pathway activation in MSC derived from two different
anatomic locations i.e. hair follicle and bone marrow, (Fig 1(a)). Furthermore,
we screened a small array of chemical inhibitors to monitor the pathways that
may control differentiation into one or more lineages.

Our results demonstrate
differential reporter activation between the two MSC sources, suggesting that
the regulatory pathways controlling differentiation may be dependent on the
source of MSC. To identify key molecules affecting MSC differentiation, several
inhibitors were selected to illustrate the effects on lineage specific reporters.
We found that inhibition of TGF-β receptor promoted adipogenesis as
evidenced by increased activity of the adiponectin promoter. Similarly, inhibition
of p38 increased the activity of both chondrogenic (SMAD2/3) and osteogenic (OCBOX)
reporters, while inhibition of TGF-β or FGF receptors down-regulated
chondrogenic or osteogenic reporters, respectively. Inhibition of the PI3
kinase and glucocorticoids readily suppressed the activity of adipogenic
pathways. Also, inhibition of JNK and ERK blocked the expression of
osteo-specific reporters such as Vitamin-D and OCBOX. Finally, we identified
novel pathways differentially regulating MSC differentiation along different
lineages. The complete results were summarized in Fig 1(b).

We developed a LVA system that
enabled efficient monitoring of gene and pathway activation in MSC
differentiation. This approach also allowed us to identify differences in the
kinetics of pathway activation between different MSC lines, despite their
similar differentiation potential. Notably, the LVA enabled screening of small
molecule libraries that identified pathways that were necessary for
differentiation along particular lineages. Our results suggest that the LVA is
a tool with great potential to enhance understanding of stem cell fate specification
as well as pathway regulation and drug discovery.