This document describes two approaches to modify stem cells to enhance their interactions with selectins and improve tissue engraftment. The first approach non-covalently couples a PSGL-1 fusion protein to mesenchymal stem cells and cardiosphere-derived cells. The second overexpresses the enzyme FUT7 to increase alpha(1,3) fucose on cell surface glycans. In vitro tests found both approaches improved selectin binding. In vivo tests in pigs found the combined approaches increased selectin-mediated cell rolling and tissue engraftment compared to unmodified cells. The work presents a strategy for enabling leukocyte-like interactions to enhance stem cell delivery efficiency.

1. Cell surface glycoengineering improves
selectin-mediated cell rolling and tethering of
stem cells
Chi Y. Lo1,2,3, Brian Weil1, Beth Palka1, Rebeccah Young1, John M. Canty Jr. 1, Sriram
Neelamegham2
1Division of Cardiovascular Medicine, 2Department of Chemical and Biological Engineering,
and 3Department of Anesthesiology; State University of New York at Buffalo
Abstract
Introduction
Both
mesenchymal
stem
cells
(MSCs)
and
cardiosphere-­‐derived
cells
(CDCs)
are
candidate
cells
currently
being
tested
in
early
phase
clinical
trials
to
determine
their
efficacy
in
trea>ng
cardiovascular
ailments.
Of
the
current
stem
cell
delivery
routes
for
cardiac
applica>ons,
vascular
injec>on
has
the
advantage
that
the
cells
have
good
access
to
nutrients
which
increases
survival
compared
with
intramyocardial
injec>on.
However,
there
is
low
cell
reten>on
in
the
heart;
thus,
we
tested
the
hypothesis
that
modifying
the
cell
surface
glycosyla>on
on
stem
cells
can
enhance
their
interac>on
to
inflammatory
sites.
Two
approaches
were
taken
to
address
the
hypothesis.
First,
the
non-­‐covalent
coupling
of
a
P-­‐selec>n
glycoprotein
ligand-­‐1
(PSGL-­‐1)
fusion
protein
to
MSCs
and
CDCs
is
performed
using
a
protein
G
lipid
conjugate
called
PPG.
Second,
we
inves>gated
the
increase
of
α(1,3)
fucose
on
the
cell
surface
glycans
by
overexpressing
the
enzyme
FUT7.
To
test
the
cell
surface
modifica>on,
cells
were
delivered
by
intracoronary
injec>on
into
pigs,
which
underwent
a
brief
10
min
ischemia.
Both
MSCs
and
CDCs
could
be
captured
onto
P-­‐selec>n
substrates
up
to
6
dyn/cm2.
FUT7
overexpression
improved
the
number
of
stem
cells
captured
onto
P-­‐
and
E-­‐
selec>n
substrates.
When
these
strategies
were
combined,
P-­‐selec>n
binding
was
further
increased.
In
vivo
experiments
demonstrated
the
approach
is
safe
and
no
adverse
events
were
noted
following
cell
injec>on.
The
current
work
presents
a
robust
strategy
for
enabling
leukocyte-­‐like
capture
and
rolling
under
constant
flow
condi>ons
to
enhance
cell
delivery
efficiency
thereby
increasing
>ssue
engra]ment.
Methods Results
Selec%n
mediated
leukocyte
tethering
and
rolling
Hypothesis:
Engineering
natural
leukocyte
adhesion
molecules
onto
MSCs
and
CDCs
can
enhance
cell
homing
to
sites
of
injury
or
inflammatory
sites
by
increasing
their
ability
to
bind
endothelial
selec?ns.
Both
mesenchymal
stem
cells
(MSCs)
and
cardiosphere-­‐derived
cells
(CDCs)
have
shown
regenera>ve
poten>al
in
the
damaged
heart.
Stem
cell
therapy
requires
targe>ng
of
cells
to
the
>ssue
repair
site
with
high
efficiency.
Two
poten>al
delivery
routes:
• Local/intramyocardial
infusion
to
damaged
>ssue
• Systemic/vascular
infusion
proximal
to
the
therapeu>c
site
Systemic
infusion
has
the
advantage
of
being
minimally
invasive,
however:
• A
large
number
of
MSCs
must
be
perfused
at
sites
of
injury
and
inflamma>on
• Engra]ment
rate
is
low
and
downstream
occlusion
may
occur
• Poten>al
non-­‐specific
distribu>on
to
other
organs
PSGL-­‐1
is
a
mucinous
glycoprotein
with
a
selec>n-­‐ligand
located
at
its
N-­‐terminus
(red
box).
19Fc
consists
of
the
first
19
amino
acids
of
mature
PSGL-­‐1
followed
by
a
human
IgG
tail.
PSGL-1:
19Fc:
Glycomics
analysis
of
O-­‐glycans
from
19Fc
and
19Fc[FUT7+]
reveal
sLeX
structure
at
m/z=1879.8
is
only
prominent
in
19Fc[FUT7+].
Approach 1:
Approach 2:
Increasing
α(1,3)
fucose
on
cell
surface
glycans
by
overexpression
of
three
fucosyltransferases
(FUT6,
FUT7,
and
FUT9)
Microfluidic
parallel
plate
flow
chamber
is
used
to
access
rolling
func>on
of
the
modified
cells.
Cells
in
Cells
out
400 µm × 100 µm × 1 cm
In vitro testing:
In vivo testing:
Acknowledgements: T32 NIH Ruth L. Kirschstein Postdoctoral Research Training Grant, NYSTEM (NY State Stem Cell initiative) and NIH (National Heart, Lung and Blood Institute)
• Cells
will
be
infused
into
the
coronary
arteries
by
a
catheter
placed
using
fluoroscopy
• Balloon
occlusion
of
the
LAD
will
cause
up-­‐regula>on
of
selec>ns
Compare
• Unmodified
CDCs
(inject
in
non-­‐ischemic
artery)
• Modified
CDCs:
FUT7-­‐DsRED
+
PPG
+
19Fc[FUT7+]
(inject
in
ischemic
artery)
Male
CDCs
were
injected
into
female
recipients
• 16
mil
unmodified
cells
(8
into
RCA,
8
into
LCX)
• 10
min
LAD
occlusion
• 30
min
reperfusion
• 8
mil
modified
cells
into
LAD
• 3
hrs
later,
>ssue
samples
were
taken
Bonow,
Robert
O.
et
al.,
“Braunwald’s
Heart
Disease.”
9th
ed.
Philadelphia,
PA:
Elsevier,
2012.
19Fc[FUT7+] coupling with PPG increases interactions with P-
selectin
Conclusion
FUT7 improves P- and E-selectin binding
Combining 19Fc[FUT7+] coupling with FUT7 overexpression
Analysis of samples from in vivo testing are still on-going…
Lo
et
al.
Biomaterials,
2013
Nov.
34(33):
8213-­‐22.
Y-­‐FISH
staining
on
male
>ssue
–
57%
posi>ve
staining
Genomic
DNA
quan>ta>ve
PCR
• Two approaches are presented that enable leukocyte-like capture to
selectins.
• These methods could be employed to increase tissue engraftment of cells
used in stem cell therapy.