Microposts As a Means to Measure the Contractile Properties of hiPSC Derived Cardiomyocytes

INTRODUCTION

Cardiomyocytes derived from
human stem cells (hSC-CMs) are an invaluable research
tool in the fight against heart disease. Recent research demonstrates that these
cells have the ability to help elucidate the mechanisms that govern heart
development and lead to heart disease, they can be used to determine the
effectiveness of candidate drugs in treating heart disease, and they have even
been shown to help reverse some of the damage caused by heart disease [1, 2]. All
of these applications can be improved
by assessing the contractility of hSC-CMs at the
single-cell level, as one of the most important functional characteristics
of a cardiomyocyte is its ability to produce contractile force. Additionally, out
of the currently available sources of human stem cells, human induced
pluripotent stem cells (hiPSCs) are very promising: the
number of cell lines that can be induced to the pluripotent state is extremely
vast, they serve as a potential source for patient-specific cardiomyocytes, and
their use is non-controversial.

In this work, we discuss the
development of an in vitro micropost
platform that is able to assess the contractile characteristics of individual hiPSC-CMs. Here, this platform was used to determine the
contractile properties of iPS-CMs of different ages, to gauge their maturation
over time. In conjunction, immunofluorescent staining was used to assess their
structural maturation. We found that, over the course of 80 days, hiPSC-CMs demonstrated significant increases in twitch
force, velocity, and power, as well as cell spread area, perimeter, and Z-band
width.

METHODS

Cell Culture

Fibroblasts from the human IMR-90 cell
line (James A. Thomson, U.Wisconsin-Madison) were
de-differentiated to the pluripotent state via treatment with transcription
factors OCT4, SOX2, NANOG, LIN28 [3]. Monolayers of these stem cells were then
differentiated into cardiomyocytes by sequential treatment of activin A and
bone morphogenetic protein 4 (BMP4) [4], supplemented with CHIR 99021 and Xav 939. These cells were cultured on Matrigel-coated
plates in RPMI medium: RPMI 1640 with L-glutamine (Gibco),
supplemented with B-27 (Gibco), and 1% penicillin/streptomyosin (Cellgro). At 20,
70, and 100 days post-differentiation, the hiPSC-CMs
were dissociated from their culture surfaces, and seeded onto arrays of
microposts in RPMI medium supplemented with %5 FBS. Following seeding, the
cells were again cultured in RPMI medium, which was henceforth exchanged every
other day.

Substrate Preparation

Arrays
of silicone microposts were fabricated by casting polydimethylsiloxane (PDMS) off
of silicon wafers with patterned SU-8 structures, onto #1 25mm round
coverslips, as previously described [5, 6]. The tips of these microposts were
coated via microcontact printing with either 50 μg/ml
of mouse laminin (Life Technologies), human fibronectin (BD Biosciences), or
human collagen IV (Millipore), while the remaining surfaces of the micropost
array were fluorescently stained with bovine serum albumin conjugated with
Alexa Fluor 594 and blocked with 0.2% Pluronic F-127 in phosphate buffer
solution [7].

Cell Analysis

One
week following cell seeding, spontaneously-beating, hiPSC-CMs
were recorded using high-speed video microscopy (Fig.
1A). A
phase contrast video was taken in the optical plane at the tips of the posts to
track the movement of individual posts during twitch contractions (Fig. 1B),
and a fluorescent image was taken at a plane where the base of the posts was in
focus to establish the reference position for each post (Fig. 1C). To determine
the deflection of each post over time, a custom-written MATLAB code was used to
find and track the movement of the posts by their centroids. The position of
the posts in each image frame was then compared to the position of the
centroids of the base of the posts (Fig. 1D). The deflection of each post over
time can then be determined with the same MATLAB code, and twitch force can be
calculated by multiplying this deflection by the bending stiffness of the
micropost (Fig. 1E). Using this same code, the contraction velocity and power
of each of the posts beneath every measured cell were determined by calculating
the change in the micropost?s deflection over time
(Fig. 1F), and from the product of the force and velocity measured at that post
over time (Fig. 1G), respectively. Finally, to obtain the total force or power
produced by an individual cell, the absolute magnitude of these entities were
summed across all the posts beneath an individual cell. Alternatively, for
velocity, each of the individual post traces was analyzed to determine the
maximum velocity that each cell was able to produce.

After
live experiments, the cells were fixed and stained for nuclei and sarcomeric α-actinin in order to image their myofibril structure
while still on the posts. Once fluorescent images were taken, NIS Elements
software was used to manually measure sarcomere lengths and Z-band widths, as
well as circularity and cell area.

RESULTS

Contractile Properties

Our analysis revealed that there
were no significant changes in passive force (Fig. 2A), force per area (Fig.
2C), or spontaneous beating frequency (Fig. 2D) for hiPSC-CMs
that were 20, 70, and 100 days post-differentiation. Alternatively, the twitch
force (Fig. 2B), velocity (Fig. 2E), and power (Fig. 2F) were all found to
significantly increase over time, in the absence of any external stimuli. However,
these quantities are still relatively small when compared to those measured for
adult cardiomyocytes.

Cytoskeletal Properties

Upon analyzing α-actinin structure of hiPSC-CMs,
we found significant increases in the spread area (Fig. 2G), perimeter (Fig. 2H),
and Z-band width (Fig. 2K), as well as a significant decrease in circularity (Fig.
2I), over time. However, there were no significant differences in sarcomere
length between any of the cell age groups (Fig. 2J), and there was an increase
in circularity between day 40 and day 70 cells. Additionally, these values are
still relatively immature in nature when compared to those found for adult
cardiomyocytes.

CONCLUSIONS

In this work, we reported on
the use of a micropost-based technology for the assessment of hiPSC-CM contractility. We demonstrated that this platform
is able to effectively measure the twitch force, velocity, and power at all
points of adhesion underneath a cell, and that is compatible with
immunofluorescent staining. Upon measuring the contractile and cytoskeletal
properties of individual iPS-CMs that were 20, 70, and 100 days post-differentiation,
we found significant changes in numerous different endpoints, consistent with cardiomyocyte
maturation. These results indicate that hiPSC-CMs observably
develop in culture over 80 days, but that these cells are still underdeveloped
when compared to adult cardiomyocytes. Thus, if hiPSC-CMs
are to be used as a model for adult human cardiomyocytes shortly after
differentiation, they require additional stimuli to attain the same level of
contractile and cytoskeletal maturation as an adult cardiomyocyte. Therefore,
future studies employing this platform will focus on determining which stimuli
are essential for enhancing hiPSC-CM maturation in
culture.

ACKNOWLEDGEMENTS

This work was funded by the
NSF Graduate Research Fellowship Program. Additionally, part of this work was conducted at the
University of Washington Microfabrication/Nanotechnology
User Facility, a member of the NSF National Nanotechnology Infrastructure
Network.

REFERENCES

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3.     Yu,
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4.     Laflamme,
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5.     Tan,
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Figure 1 The
twitch characteristics of a single hiPSC-CM
is determined by taking a high-speed video of the tips of the posts and
a reference image of the base of the posts (A). This video is taken in the
phase contrast setting (B), while the reference image is taken in the red
fluorescent channel (C). A custom MATLAB code is then used to determine the
location of each post?s centroid in the reference plane (red dot), as well as
it is used to track the location of the post?s centroid in the video plane
(blue dot) (D). The difference in location between these two centroids over
time gives the deflection of a post over multiple twitch events, which can be
multiplied by the post stiffness to yield the twitch force (E). This deflection
data can then be used to determine the twitch velocity (F), and power (G) of
that post. Scale bars represent 6 μm.

Figure 2 Upon
investigating the structural and contractile maturation of 20, 70, and 100 day
old hiPSC-CMs, we found no statistical changes in
passive force (A), force per area (C), frequency (D), or sarcomere length (J).
However, significant differences in twitch force (B), upstroke (black) and
relaxation (grey) velocity (E), upstroke (black) and relaxation (grey) power
(F), spread area (G), cell perimeter (H), circularity (I), sarcomere length
(J), and Z-band width (K) were found. A representative hiPSC-CM
is shown for reference (L). Scale bar indicated 6 μm.