Engler, Adam J.

Taylor-Weiner, Hermes

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Extracellular Matrix Proteins are Necessary for Mouse Embryonic Stem Cell Differentiation and May Guide Stem Cell Fate 

Wednesday, February 29, 2012 719aexhibit a local hexagonal pack-
ing and evolve from disordered
agregates to spooled then en-
tangled straight bundles as the
rigidity of lipid-coated polyelec-
trolytes increases. These assem-
blies may constitute a generic
route for interfacing polyelectro-
lytes to living cells like in gene
delivery.
3643-Pos Board B504
Structure, Assembly and Mechanical Properties of DNA Nanoparticles
Condensed with Peim
Preethi L. Chandran1, Emilios K. Dimitriadis2, Ferenc Horkay3.
1National Institutes of Health, Bethesda, MD, USA, 2Laboratory of
Bioengineering and Physical Science, NIBIB, National Institutes of Health,
Bethesda, MD, USA, 3Section on Tissue Biophysics and Biomimetics,
NICHD,National Institutes of Health, Bethesda, MD, USA.
DNA is an anionic polyelectrolyte, which occupies a large volume in salt free
solution due to the coulomb repulsion between the charged groups. In the pres-
ence of polymer cations, DNA condenses into nanoparticles. DNA nanopar-
ticles have generated a lot interest as a preferred vehicle for delivering
therapeutic DNA in gene therapy. The efficiency of gene delivery is determined
by stability and compactness of the particles. However, not much is known
about the organization of DNA within the nanoparticles. Large polymer cations
condense DNA rapidly, with no distinct intermediate stages that could give in-
sight into the arrangement of DNA. In our work, we modulate the DNA length
to slow down nanoparticle formation; and, by imaging with Atomic Force Mi-
croscopy, reconstruct stages in the particle assembly. The polymer cation used
was polyethyleneimine modified with sugar residues (PEIm). The cation:base
pair ratio was ~60. The DNA is found to be arranged within the nanoparticle
as an inter-weaving network of long fiber condensates. The fiber condensates
form from DNA folding along its length, and appear to be the unit of DNA or-
ganization within the particle. The fiber-condensate network is highly deform-
able, having as much as 95% water content. Nano-indentation experiments
suggest that the nanoparticles have a hollow sphere architecture.
3644-Pos Board B505
Extracellular Matrix Proteins are Necessary for Mouse Embryonic Stem
Cell Differentiation and May Guide Stem Cell Fate
Hermes Taylor-Weiner, Adam J. Engler.
University of California, San Diego, La Jolla, CA, USA.
While in vitro differentiation protocols often rely exclusively on soluble growth
factors to direct mouse embryonic stem cell (mESC) fate, the ESC niche con-
tains both soluble factors and fibrillar extracellular matrix (ECM) proteins, in-
cluding fibronectin (FN). Moreover, some of the soluble factors used to guide
differentiation (e.g. Activin A) are known to increase the expression of ECM
proteins, though the functional importance of this change is not well under-
stood. We examined whether ECM proteins were necessary for promoting
and directing mESC differentiation. mESCs, grown as embryoid bodies under
differentiating conditions in the absence of FN, maintained expression of the
pluripotency marker, Nanog. The embryoid bodies also showed a spatiotempo-
ral correlation between expression of FN and GATA4, a marker for differenti-
ation. When differentiated on a gelatin substrate, mESCs create a fibrillar ECM
containing fibronectin and laminin components, while the presence of leukemia
inhibitory factor (LIF), a maintainer of mESC pluripotency, inhibits the pro-
duction of this fibrillar matrix. Ongoing work is investigating how the compo-
sition of this cell-derived ECM changes when soluble growth factors are used to
guide ESC fate and whether these changes are necessary to efficiently direct
differentiation. Together these data imply that FN is necessary for mESC dif-
ferentiation and that the extracellular matrix may be an important director of
stem cell fate.
3645-Pos Board B506
Investigating the Effects of Dynamic External Stimuli on Single Cell
Fitness and Gene Expression in Escherichia Coli
Eric M. Johnson-Chavarria, Utsav Agrawal, Melikhan Tanyeri,
Charles M. Schroeder.
University of Illinois, Urbana-Champaign, IL, USA.
In this work, we study the bacterial cellular response to time-dependent exter-
nal stimuli in single living cells. We developed a microfluidic platform for sin-
gle cell analysis that allows for dynamic control of well-defined environmental
growth conditions and culture media. Using this platform, we studied the effect
of small molecule inducers on gene expression in the lac operon using fluores-
cent reporter proteins and cell growth rates as a proxy of cellular fitness. Weapplied temporally varying inducer concentrations by translating single cells
between two adjacent fluid streams containing either growth medium or growth
medium and inducer. We observed that single cell gene expression depends on
growth rate and frequency of exposure to inducer concentrations. Single cell
induction experiments are compared to control experiments with and without
continuous fluid flow in microfluidic channels. For these experiments, single
cell analysis is facilitated by a microfluidic-based hydrodynamic trap recently
developed in our lab. The hydrodynamic trap enables confinement and manip-
ulation of single cells in free solution using the sole action of fluid flow. Auto-
mated feedback control is integrated into the device using an ‘‘on-chip’’ valve,
which allows for precise confinement of cells in free solution. Using this de-
vice, cells are confined at a fluid stagnation point generated in a cross-slot mi-
crofluidic geometry, thereby enabling non-perturbative trapping of cells for
long time scales. Using optical microscopy, we observe single Escherichia
coli cells growing and dividing both at room temperature and at 370C, and
cell division rates in the microfluidic trap compare favorably to the growth rates
of E. coli measured in bulk studies. We anticipate that the microfluidic-based
trap is an ideal platform to study cellular regulation and gene network dynamics
of single living cells in free solution.
3646-Pos Board B507
Infrared Light Excites Cells via Transient Changes in Membrane Electri-
cal Capacitance
Mikhail G. Shapiro1,2, Kazuaki Homma3, Sebastian Villarreal2,
Claus-Peter Richter3, Francisco Bezanilla2.
1UC Berkeley, Berkeley, CA, USA, 2University of Chicago, Chicago,
IL, USA, 3Northwestern University, Chicago, IL, USA.
Control of excitable cells using optical technologies such as optogenetics has
enabled important advances in neuroscience and the development of clinical
applications. Most existing methods of optical control require the use of genetic
or chemical sensitizers that enable light to alter the ionic conductance of cell
membranes. By contrast, infrared (IR) light of wavelengths > 1.5mm has
been shown in vivo to excite neural and muscle tissue without any pre-
treatment. Unfortunately, the mechanism of IR stimulation is unknown.
Here, we describe how IR light excites cells by transiently altering their mem-
brane electrical capacitance. Our data from voltage clamped Xenopus laevis
oocytes, mammalian cells and artificial lipid bilayers shows that IR energy ab-
sorbed by water produces a rapid local increase in temperature at the cell mem-
brane, transiently increasing its electrical capacitance, and generating
depolarizing currents. Correspondingly, under current clamp conditions, IR
pulses produce rapid changes in membrane potential. This unexpected mecha-
nism is fully reversible and requires only the most basic properties of cell mem-
branes. Changes in capacitance were verified by direct measurement in
mammalian cells and artificial bilayers, and are consistent with a classical the-
oretical description of cell membranes as coupled double-layer capacitors. In
shedding light on the mechanism of IR stimulation, our findings point to this
technology’s unique generality as a means to control excitable cells, and raise
questions about other thermal phenomena that may meaningfully affect mem-
brane electrostatics. Supported by the NIH: GM030376 and DC011481-01A1.
3647-Pos Board B508
Mechanical Derivation of Functional Myotubes from Adipose-Derived
Stem Cells
Yu Suk Choi, Ludovic Vincent, Andrew Lee, Marek Dobke, Adam Engler.
University of California, San Diego, La Jolla, CA, USA.
In recent years, ECM stiffness and resulting cell contractility have been iden-
tified as potent stem cell differentiation regulators. Successful stem cell-based
therapies will require acclimating cells to the abnormally stiff ECM of muscu-
lar dystrophy while inducing and/or maintaining myogenesis, fusion, and dys-
trophin delivery. Here we directly compare ASC to BMSC stiffness
responsiveness and show myotube formation derived from ASCs on matrices
that mimic skeletal muscle. ASCs are shown here to not just simply reflect
the qualitative stiffness sensitivity of bone-marrow-derived stem cells
(BMSCs) but to exceed BMSC myogenic capacity (40-fold higher myogenic
marker expression on myogenic stiffness), expressing the appropriate temporal
sequence of muscle transcriptional regulators on muscle-mimicking extracellu-
lar matrix in a focal adhesion- and contractility-dependent manner. 2% of ASCs
formed multi-nucleated myotubes with a continuous cytoskeleton (10-fold
higher than chemical induction) that was not due to misdirected cell division;
microtubule depolymerization severed myotubes, but after washout, ASCs re-
fused at a rate similar to pretreated values. BMSCs never underwent stiffness-
mediated fusion. ASC-derived myotubes, when replated onto non-permissive
stiff matrix, maintain their fused state. Fusion frequency was increased by
a contractile agonist, lysophosphatidic acid and decreased by a myosin inhibi-
tor, blebbistatin. ASCs generated higher tangential force than BMSCs and


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Extracellular Matrix Proteins are Necessary for Mouse Embryonic Stem Cell Differentiation and May Guide Stem Cell Fate

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