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2014 CMBEC37 Conference
Vancouver, BC
May 21 – 23, 2014
NEURONAL DIFFERENTIATION OF HUMAN INDUCED PLURIPOTENT STEM
CELLS SEEDED ON MELT ELECTROSPUN MICROFIBERS
Nima Khadem Mohtaram1, Junghyuk Ko1, Craig King2, Amy Montgomery1, Lin
Sun3, Rishi Vasandani1, Martin Byung-Guk Jun1 and Stephanie M. Willerth 1,3,4,*
1 Department of Mechanical Engineering, University of Victoria. PO Box 1700,
STN CSC, Victoria, BC V8W 2Y2, Canada
2Department of Biomedical Engineering, University of Victoria. PO Box 1700,
STN CSC, Victoria, BC V8W 2Y2, Canada
3 Department of Biochemistry and Molecular Biology, University of Victoria, V8P
5C2, Victoria, BC, Canada
4 Division of Medical Sciences, University of Victoria. PO Box 1700, STN CSC,
Victoria, BC V8W 2Y2, Canada
*Correspondence to: Stephanie M. Willerth
E-mail: [email protected]
INTRODUCTION
Human induced pluripotent stem cells
(iPSCs) were first derived from human
fibroblasts by the Nobel Prize winner Yamanaka
and his colleagues [1]. Human iPSCs have two
prominent properties: pluripotency and the
ability to self-renew. Human iPSCs are an
alternative to human embryonic stem cells
(ESCs) since reprogramming adult cells can be
used to for producing patient specific cell lines
and avoids the bioethical issues related to
human ESCs [2, 3]. One of the major
challenges when differentiating human iPSCs is
how to control this process to produce the
desired cell phenotypes. To obtain mature
neurons, iPSCs are treated with chemical cues
such as growth factors and also physical cues
to stimulate the differentiation of stem cells into
neurons [4, 5]. In this work, we investigate
how the architecture of melt electrospun
scaffolds influences the differentiation of human
iPSCs into neurons and their resulting
alignment. This combination of hiPSC-dervied
neurons seeded on such electrospun scaffolds
could be used for neural tissue engineering
applications.
MATERIALS AND METHODS
Fabrication and characterization of scaffolds
Poly (Ɛ-caprolactone) (PCL), (Mn: 45,000)
was purchased from Sigma Aldrich Chemical
Co, USA. A custom-made melt electrospinning
setup was used to fabricate PCL microfiber
scaffolds with different architectures including
loop mesh and biaxial mesh[6]. Microstructure
characterization was performed using a Hitachi
S-4800 FE scanning electron microscopy (SEM)
machine. Melt electrospun scaffolds were
carbon coated prior to imaging.
Human iPSC Culture
All reagents used were purchased from
STEMCELL
Technologies
unless
otherwise
stated. Human iPSCs were cultured on a
Vitronectin XF™ matrix and in the presence of
TeSR™-E8™ media. The human iPSCs were
incubated at a 5% carbon dioxide (CO2) level
and a temperature of 37ºC to mimic body
conditions. To begin the differentiation process,
confluent human iPSC colonies were dissociated
into a single cell solution using Gentle Cell
Dissociation Reagent. The single cell solution
was distributed into an Aggrewell™ 800 plate.
These plates contain microwells with a diameter
of 800 µm that uses gravity to form consistent,
spherical aggregates from the hiPSCs. At 90%
confluence
the
colonies
dissociate
into
Proceedings of the 37th Canadian Medical and Biological Engineering Conference – 2014
approximately
6
million
cells
yielding
approximately 20,000 cells per aggregate. To
induce neural differentiation, these cells were
cultured in the presence of STEMdiff™ Neural
Induction Medium (NIM) for 5 days. 2 mL of
NIM was added to each well with 1.5 mL
changed daily. NIM directs the differentiation of
the hiPSCs into neural progenitor cells.
fibers. The biaxial mesh scaffolds consisted of
20 layers of straight fibers. A layer of horizontal
fibers were laid on top of a layer of vertical
fibers; this was repeated ten times. Both
topographies can be seen in Figure 1.
Neural progenitor cell seeding onto scaffolds
After 5 days of formation, neural aggreagtes
containing neural progenitors cells were seeded
onto loop mesh and biaxial mesh scaffolds.
Approximately four neural aggregates were
seeded into each well of the 6 well plates
containing loop mesh or biaxial mesh scaffolds.
The neural aggregates were cultured in NIM for
12 days.
Cell viability and immunocytochemistry analysis
The viability of neural aggregates seeded on
the PCL microfiber scaffolds was analyzed
qualitatively after 12 days by using a
LIVE/DEAD®
Viability/Cytotoxicity
Kit
(Invitrogen). The kit contains a stain for
viability, calcein AM, which is enzymatically
converted to green fluorescing calcein by the
naturally present intracellular esterase activity
in live cells, and a stain for cytotoxicy, ethidium
homodimer-1, which fluoresces red upon
binding to nucleic acids accessed through the
ruptured cell membranes of dead cells. The
details are given in our previous work [6].
Neuronal
differentiation
of
hiPSCs
was
qualitatively assessed by immunocytochemistry
targeting the neuron-specific protein β-IIItubulin as previously described [6]. Images
were captured for green and blue fluorescence.
Images were overlaid at layer opacity of 50%.
Higher magnification fluorescent images were
acquired on a LEICA 3000B inverted microscope
using an X-cite series 120Q fluorescent light
source (Lumen Dynamics) coupled to a Retiga
2000R fast cooled mono 12-bit camera (Qimaging).
RESULTS AND DISCUSSION
Topographical characterization of scaffolds
The loop mesh scaffolds consisted of two
layers of looped fibers. A layer of horizontal
fibers were laid on top of a layer of vertical
Figure 1. : Scanning electron microscope images of loop
mesh and biaxial mesh melt electrospun scaffolds.
Effect of scaffold architecture
differentiation of hiPSCs
on
neuronal
Neural aggregates derived from human
iPSCs were seeded on loop mesh and biaxial
mesh scaffolds for 12 days. The differentiation
of the seeded cells was qualitatively analyzed
with
fluorescent
microscopy
and
immunocytochemistry.
Immunocytochemistry
uses primary antibodies and conjugated
secondary antibodies to target β-III-tubulin, a
neuron specific protein. Bright field, Live/Dead,
and immunocytochemistry images of cells
seeded on loop mesh and biaxial mesh scaffolds
can be seen in Figures 2 and 3, respectively.
Higher magnification bright field images of cells
seeded on loop mesh and biaxial mesh scaffolds
can be seen as well. It can be seen in the bright
field images that both scaffold topographies
support cell adhesion and cell migration. The
cells have started to migrate outward from the
spherical neural aggregates along the straight
or looped fibers, depending on the scaffold
morphology. The cells have also started to
migrate between the pores of the scaffold and
fill the loop and rectangular structures. It
appears that the smaller pores are easier for
the cells to migrate and fill. The Live/Dead
images
demonstrate
that
both
scaffold
topographies are viable substrates for human
iPSCs because the majority of the seeded cells
are alive.
A small amount of cell death was observed;
however the dead (red fluorescing) cells
present may be due to the media. The cells
metabolize the nutrients in the media and
release waste by-products, which lowers the
2014 CMBEC37 Conference
Vancouver, BC
May 21 – 23, 2014
levels of nutrients and raises the level
of toxins. This could be addressed by changing
the media during the 12 day seeding period.
After comparing the immunocytochemistry
images to their corresponding bright field
images it is apparent that not all the cells
fluoresce. It appears that the neurons have
grown along the scaffold fibers and dense cell
areas which have the most support. The cells
that are not fluorescing appear to be the outer
cell growth in the porous areas between the
scaffold.
Figure 2. : Neural progenitor cells seeded on loop mesh
scaffolds after 12 days. (A) is a bright field image
corresponding to its Live/Dead staining counterpart (B). (C)
is a bright field image corresponding to its
immunocytochemistry staining counterpart (D). (E) is
higher magnification image corresponding to the white
rectangle in (D). The scale bar is 800 μm.
Figure 3. Neural progenitor cells seeded on biaxial mesh
scaffolds after 12 days. (A) is a bright field image
corresponding to its Live/Dead staining counterpart (B). (C)
is a bright field image corresponding to its
immunocytochemistry staining counterpart (D). (E) is an
higher magnification image corresponding to the white
rectangle in (D). The scale bar is 800 μm.
CONCLUSION
For the first time, we have shown that
human iPSCs can differentiate into neurons,
while seeded onto melt electrospun fibrous
scaffolds. Particularly, the physical cues of
electrospun microfiber scaffolds on the viability
and neuronal differentiation of human iPSCs
were investigated. The Live/Dead images
proved that both microfiber scaffolds were
viable substrates to neural progenitor cells due
to the high ratio of live cells over dead cells.
Lastly, both microfiber scaffolds fostered
Proceedings of the 37th Canadian Medical and Biological Engineering Conference – 2014
neuronal differentiation as the seeded cells
expressed the neuron specific protein β-IIItubulin. A novel combination of the microfibers
with hiPSCs would be introduced as a promising
approach
for
neural
tissue
engineering
applications.
ACKNOWLEGMENTS
The authors would like to acknowledge
funding support from Natural Sciences and
Engineering
Research
Council
(NSERC)
Discovery Grant Program, Canadian Foundation
for Innovation, British Columbia Knowledge
Development
Fund
and
the
Advanced
Microscopy Facility at the University of Victoria.
REFERENCES
1.
Takahashi, K., et al., Induction of pluripotent stem
cells from adult human fibroblasts by defined
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2.
Willerth, S.M., Neural tissue engineering using
embryonic and induced pluripotent stem cells.
Stem Cell Research & Therapy, 2011. 2.
3.
Yu, J.Y., et al., Induced pluripotent stem cell lines
derived from human somatic cells. Science, 2007.
318(5858): p. 1917-1920.
4.
Lopez-Gonzalez, R. and I. Velasco, Therapeutic
potential of motor neurons differentiated from
embryonic stem cells and induced pluripotent stem
cells. Archives of medical research, 2012. 43(1):
p. 1-10.
5.
Pan, F., et al., Topographic effect on human
induced pluripotent stem cells differentiation
towards neuronal lineage. Biomaterials, 2013.
34(33): p. 8131-9.
6.
Ko, J., et al., Fabrication of poly (epsiloncaprolactone) microfiber scaffolds with varying
topography and mechanical properties for stem
cell-based tissue engineering applications. Journal
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25(1): p. 1-17.