StemAdhere™ and StemAdhere™ XF: Defined and Xeno-free Substrates for Stem Cell Culture
Scott A. Monsma, Bradley H. Garcia II, and Andrea Marty. Primorigen Biosciences, Madison, WI, USA, 53719
Abstract
Morphology
In order to establish clinically adaptable methods for
therapeutic applications of stem cells, there is increasing
interest in stem cell expansion, propagation, and
differentiation under completely defined and xenobioticfree conditions. Nagaoka et al. have previously shown that
E-Cadherin fused to mouse IgG Fc domain is a suitable substrate
for iPS and hES growth and maintenance of pluripotency1.
Extending this work, Primorigen has developed StemAdhere™
as a completely defined substrate of completely human
sequence useful for routine propagation and expansion of stem
cells. StemAdhere can be handled at room temperature, and is
compatible with gentle, non-enzymatic cell release methods.
Growth and pluripotency of human iPSCs on StemAdhere
has been characterized in 3 commercially available defined
and/or xeno-free stem cell media, and extended passages are
currently underway.
To enable completely defined and xeno-free culture systems,
StemAdhere™ XF has been developed. StemAdhere™ XF is
in testing with commercially available xeno-free media and
initial results will be presented.
StemAdhere™
day 1
StemAdhere™ XF
BD Matrigel™
mTeSR®1
mTeSR®1
TeSR™2
mTeSR®1
TeSR™2
TeSR™2 day 4
mTeSR®1
TeSR™2
mTeSR®1 day 4
day 2
day 4
Mechanisms of Adhesion
EC1
EC2
EC3
Cellular
E-Cadherin
Proliferative and
anti-apoptotic
signaling
Clustering
Ca2+
EC5
Ca2+ Ca2+
Immobilization
Domain
Immobilized
StemAdhere™
Polystyrene surface (hydrophobic uncharged)
StemAdhere takes advantage of the homophilic, calcium
dependent adhesion molecule E-Cadherin. E-Cadherin mediates
cell-cell adhesion in blastocyst embryos and within colonies of
iPS or ES cells. Expression of E-Cadherin is lost early in normal
development6 (during the Epithelial-Mesenchymal Transition
or EMT), and E-Cadherin is down-regulated during directed
differentiation into ectoderm, endoderm or mesoderm.
Degradation of E-Cadherin can occur during single-cell passaging
resulting in rapid cell death; ROCK inhibitors such as Y-276323 or
thiazovivin4 can prevent degradation and cell death.
Proliferation
Pluripotency Markers
120
mTeSR1 Pluripotency
120
100
100
80
80
% (+) Cells
--S--S--
% (+) Cells
Ca2+
Ca2+
↓ EGFR/ERK
↑ PI3-K/AKT 5
EC4
Ca2+ Ca2+
EC4
Ca2+
Ca2+
60
40
20
0
Oct4+
BD Matrigel™
SSEA4+
StemAdhere™
TeSR2 Pluripotency
1.8
1.6
60
40
20
0
Oct4+
BD Matrigel™
StemAdhere™
SSEA4+
iPS cells were grown on Matrigel or StemAdhere for 5 passages
in mTeSR1 or TeSR2 medium. Cells were harvested and analysed
by flow cytometry for pluripotency markers Oct4 and SSEA-4.
(iPS line C2 courtesy of S. Duncan).
Summary
• StemAdhere™ and StemAdhere™ XF support proliferation
and pluripotency in both mTeSR1 and TeSR2.
• After adaptation to StemAdhere, proliferation rates are
comparable to those seen with BD Matrigel.
• Cells/colonies spread out more on StemAdhere and
StemAdhere XF than on BD Matrigel or feeders,
suggesting tighter adhesion to the substrate.
ECM substrates such as BD Matrigel™ offer a variable number
of adhesion partners including collagen, laminin, vitronectin,
and nidogen. Adhesion occurs by interactions with plasmamembrane embedded integrins.
2
Cells/well (million)
EC1
EC2
Ca2+ Ca2+
EC3
Ca2+
iPS cells and colonies on StemAdhere adopt a flattened, spread out morphology.
Cell-cell boundaries are distinct and colony edges may appear more ragged.
Cells behaved identically on StemAdhere and StemAdhere XF, in both mTeSR®1
and TeSR™2 (Stem Cell Technologies). Photos represent iPS line K3 (courtesy of
S. Duncan) at p59 at day 1, 2 and 4 after passaging with Cell Release Buffer (nonenzymatic).
Ca2+
Ca2+ Ca2+
Ca2+
EC5
E-Cadherin Ectodomain
Adhesion
Domain
1.4
SA mTeSR1
SA XF mTeSR1
MG mTeSR1
SA TeSR2
SA XF TeSR2
MG TeSR2
1.2
1
0.8
0.6
0.4
0.2
0
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
Day
iPS cells were grown on Matrigel, StemAdhere or StemAdhere XF
in mTeSR1 or TeSR2 medium. Cells harvested from duplicate wells
were counted at each passage (every 3 days) and sister wells were
passaged onto fresh plates at 0.2 x 106 cells/well in 6-well plates.
(iPS line K3 courtesy of S. Duncan).
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