1. Combat Casualty Care
P R O T E C T
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INSTITU
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OF SURGICAL RE
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EARCH
Effect of Embryoid Body Size on Differentiation of iPS Cells into
Neuroectodermal-Lineage Ocular Cells
O.A. Creasey1, A. Muñiz2, M.L. Plamper2, H-C.H. Wang2
1Summer Intern, Pittsburgh Tissue Engineering Initiative, 2United States Army Institute of Surgical Research
Department of Ocular Trauma, Fort Sam Houston, TX 78234-6315
Results (Continued)Abstract
Cell death in ocular tissues such as the retina and retinal pigment
epithelium (RPE) due to trauma, disease, and aging is a major cause of
blindness. Potential therapies to halt or reverse vision loss may require
replacement of damaged cells with stem cell derived tissues. Current
differentiation methods for human induced pluripotent stem cells (hiPS
cells) and human embryonic stem cells have allowed the derivation of
neuroectodermal cells such as retinal neurons and RPE cells [1, 2, 3], but
only at low efficiencies. Higher efficiency differentiation methods are
essential if these cell-based therapies are to be applied to human patients.
Past research has shown that embryoid body (EB) size can affect the
efficiency with which stem cells differentiate into germ layers [4]. This
study investigates the effect of EB size on efficiency of differentiation of
iPS cells into neuroectodermal cells.
In preparation for embryoid body formation, hiPS (IMR-90-1) cells were
cultured on matrigel. Different sized EBs were formed using AggreWell
plates according to manufacturer’s instructions. The EBs were
subsequently plated on matrigel in six-well plates and fed with
differentiation medium. At Day 6, Day 17, and Day 29 RNA was collected
from the EBs and RT-PCR was performed to determine expression of the
PAX6 gene, a marker specific to neuroectodermal cells. In addition, RT-
PCR was run on the Day 17 and Day 29 RNA to determine expression of
the MiTF gene, a marker specific to development of ocular cells of
neuroectodermal lineage [3, 5].
PAX6 was expressed in cells from all four EB sizes at Day 6, Day 17, and
Day 29. At Day 6, PAX6 expression increased with EB size. At both Day
17 and Day 29, EBs containing 500 and 3000 cells expressed more PAX6
than EBs containing 200 or 15000 cells. MiTF was also expressed at Day
17 and Day 29 in all four EB sizes. At Day 17 expression was higher in
the 200, 500, and 3000 EBs than in the 15000 cell EBs. At Day 29
expression was highest in the 500 cell EBs.
These results show that gene expression differs between the cells of
different EB sizes. They also show that although larger EBs have the
highest expression of PAX6 at early timepoints and therefore could be
optimal for differentiation into neuroectodermal cells, medium-sized EBs
(500 and 3000 cells) are best for differentiation of neuroectodermal-
lineage ocular cells as indicated by co-expression of PAX6 and MiTF at
later timepoints. We speculate that the loss of PAX6 expression in larger
EBs at later timepoints is due to cell-cell interactions between increasingly
confluent cells.
Objectives
The objective of this study is to determine the effect of embryoid body
size on differentiation of neuroectodermal cells from human iPS cells.
Methods
Culture and Maintenance of iPS cell cultures: Human iPS (IMR-90-1
cells) from WiCell were cultured on matrigel (BD Biosciences) coated six
well plates and maintained in mTeSR1 media (Stem Cell Technologies).
The medium was changed daily until cells were ready for passage (approx
4-5 days).
Conclusions
Acknowledgements
We would like to acknowledge Joan Schanck and LaShon Jackson of PTEI for
coordinating support and thank PTEI for making this research opportunity available.
We also acknowledge Dr. David Baer and Dr. Robert Christy of the United States
Army Institute of Surgical Research and all of the other USAISR and BAMC staff
for their help in coordinating this opportunity.
• EB size affects the differentiation pathway of hiPS cells.
• Larger EB sizes encourage expression of the neuroectodermal marker
PAX6 at early timepoints.
• Medium EB sizes, especially the 500 cell EBs, encourage expression of
PAX6 and MiTF at later timepoints.
• Cell-cell interactions between increasingly confluent cells could affect
expression of PAX6 at later timepoints.
• In the future EB size will be used to control hiPS cell differentiation.
References
[1] Buchholz, D. E., S. T. Hikita, et al. (2009). "Derivation of functional retinal
pigmented epithelium from induced pluripotent stem cells." Stem Cells 27(10):
2427-2434.
[2] Idelson, M., R. Alper, et al. (2009). "Directed differentiation of human
embryonic stem cells into functional retinal pigment epithelium cells." Cell Stem
Cell 5(4): 396-408.
[3] Meyer, J. S., R. L. Shearer, et al. (2009). "Modeling early retinal development
with human embryonic and induced pluripotent stem cells." Proc Natl Acad Sci U S
A 106(39): 16698-16703.
[4] Bauwens, C. L., R. Peerani, et al. (2008). "Control of human embryonic stem
cell colony and aggregate size heterogeneity influences differentiation trajectories."
Stem Cells 26(9): 2300-2310.
[5] Vaajasaari, H., T. Ilmarinen, et al. (2011). "Toward the defined and xeno-free
differentiation of functional human pluripotent stem cell-derived retinal pigment
epithelial cells." Mol Vis 17: 558-575.
Disclaimer: The opinions or assertions contained herein are the private views of the
author and are not to be construed as official or as reflecting the views of the
Department of the Army or the Department of Defense.
Methods (Continued)
Results
Figure 1: iPS cells in Culture. The cells have a small & round
phenotype, a characteristic of pluripotent stem cells. (magnification
100x) IPS cells were cultured on matrigel in mTeSR1 at 37˚C and 5%
CO2.
Figure 2: EB formation in AggreWell plates. Each microwell
contains A. 200 cells, B. 500 Cells, C. 3000 Cells. Cells were
incubated 24 hours at 37º C and 5% CO2 for the formation of EBs.
(magnification 100x)
Figure 3: Different sized EB-derived colonies on Day 2 of
differentiation. A. 200 cells, B. 500 Cells, C. 3000 Cells, D. 15,000
Cells. Cells were incubated for two days at 37º C and 5% CO2 in
differentiation medium after the EBs were plated on matrigel-coated
plates. (magnification 20x)
Figure 4: Expression of PAX6 and MiTF at Day 17 of
differentiation. All four EB sizes showed expression of PAX6. The
500 and 3000 cell EBs show higher PAX6 expression than that of the
200 and 15000 cell EBs..All four EB sizes showed expression of
MiTF with higher levels in the 200, 500, and 3000 cell EBs. The
15000 EBs showed the lowest levels of MiTF. The error bars show
standard error.
A B
C D
Results (Continued)
BA
C -1.20
-0.88
-0.87
-1.19
0.00
-1.01
-0.97 -0.94
-1.45
0.00
-1.6
-1.4
-1.2
-1
-0.8
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0
0.2
200 500 3000 15000
Log10(RelativeQuantitation)
EB Size
Expression of PAX6 and MiTF at Day 17
PAX6
MiTF
-0.49
-0.20
0.05
0.29
0.00
-0.6
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0
0.1
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200 500 3000 15000
Log10(RelativeQuantitation)
EB Size
Expression of PAX6 at Day 6
PAX6
Figure 4: Expression of PAX6 at Day 6 of differentiation. All four
EB sizes showed expression of PAX6 and expression increased with
increasing EB size. The 200 and 500 cell EBs had a lower expression
of PAX6 than ARPE-19 cells while the 15000 cell EBs had a higher
expression of PAX6 than ARPE-19 cells. Expression of PAX6 in the
3000 EBs was roughly the same as in ARPE-19 cells. The error bars
show standard error.
Formation of Embryoid bodies: Embryoid bodies (EBs) consisting of
200, 500, 3000 and 15000 cells were prepared using AggreWell 400 &
800 plates. Briefly, an iPS single cell suspension was prepared in
AggreWell Medium and seeded in AggreWell plates at a density that
would yield the desired number of cells per microwell. The AggreWell
plates were then centrifuged at 100 x g for 3 minutes to capture the cells in
the microwells . The plates were then incubated at 37 ºC and 5% CO2 for
24 hours to allow formation of EBs. The EBs were then collected and
seeded in matrigel coated six well plates in AggreWell Medium for 24
hours. To initiate the differentiation protocol the AggreWell Medium was
then replaced with differentiation medium consisting of 10% Knockout
serum, 0.1mM β-Mercaptoethanol, 0.1 mM nonessential amino acids, 2.0
mM Glutamine and 10 µg/ml gentamicyn in DMEM/F12.
RNA Extraction and RT-PCR: RNA was extracted from the cell cultures
using an RNeasy Plus Mini Kit (Qiagen). Briefly, cells were either lysed
directly in the six-well plates using Buffer RLT Plus (Qiagen) or removed
from the plate with accutase and re-suspended in Buffer RLT Plus after
washing. The lysate was then homogenized using QIAshredder spin
columns (Qiagen) and the RNA was extracted using RNeasy Plus Mini Kit
protocol. The RNA was translated to cDNA using a High Capacity
RNA-to-cDNA Kit (Applied Biosystems) and then PCR was run using
Taqman Gene Expression Assays for PAX6 and MiTF on a 7300 Real
Time PCR System (Applied Biosystems). 18S served as the internal
control. The data was then analyzed using the ΔΔ CT method. The RQ of
PAX6 and MiTF in ARPE19 cells are made to equal to 1 and are used as
the baseline for analysis. The data is plotted as the log10 of the RQ.
-1.12
-0.51
-0.73
-1.12
0.00
-1.01
-0.66
-0.90
-0.82
0.00
-1.2
-1
-0.8
-0.6
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0
200 500 3000 15000
Log10(RelativeQuantitation)
EB Size
Expression of PAX6 and MiTF at Day 29
PAX6
MiTF
Figure 5: Expression of PAX6 and MiTF at Day 29 of
differentiation. All four EB sizes showed expression of PAX6 and
MiTF. The highest PAX6 expression is seen in the 500 cell EBs with
somewhat lower expression in the 3000 cell EBs and even lower
expression in the 200 and 15000 cell EBs. The highest expression of
MiTF was also seen in the 500 cell EBs with lower expression in the
3000 and 15000 cell EBs and the lowest expression in the 200 cell
EBs. The error bars show standard error.