Table 1.
Primer list.
Table 2.
Mechanical properties for aligned PLGA nanofiber scaffolds.
Fig 1.
Cells seeded on A) Flat surface plate and B) Aligned nanofiber characterized by cell anisotropic index (CAI). The hiPSC-CMs were stained with phalloidin dye (Red) to determine the alignment of seeded cells. CAI was significantly increased in the aligned nanofiber group.
Fig 2.
Analysis of gap junctions in hiPSC-CMs by confocal microscopy in cells seeded on flat plate and aligned nanofiber.
The gap junctions in hiPSC-CMs was evaluated by CX-43 immunostaining of human cardiomyocytes seeded on flat surface versus aligned-nanofiber coated coverslips. A) Flat plate, CX-43 (Red), SAA (Green) and DAPI (Blue); B) Aligned nanofiber, CX-43 (Red), SAA (Green) and DAPI (Blue); C) Quantification of gap junctions (% CX-43 positive area fraction per cell) in hiPSC-CMs seeded on flat plate and aligned nanofiber. There were no significant differences between the two groups. All values expressed as mean ± SD (n = 4/group).
Fig 3.
Confocal Imaging of mitochondria.
Mito-tracker red staining shows alignment of Human iPSC-Cardiomyocytes seeded on A) Flat surface vs B) Aligned-nanofiber coated coverslips (32x & 200x). C) TEM imaging showing comparison of mitochondrial morphology and arrangement of hiPSC-CMs seeded on flat plate versus aligned nanofiber groups.
Fig 4.
SEM imaging of Human iPSC-Cardiomyocytes.
Cells seeded on A) Flat surface vs B) Aligned-nanofiber coated coverslips (600x & 1200x) show different cell morphology.
Fig 5.
TEM imaging of Human iPSC-Cardiomyocytes.
Cells seeded on Flat surface A) nucleus, B) mitochondria, C) myofibrils into sarcomere. Cells seeded on aligned nanofiber D) nucleus, E) mitochondria and F) myofibrils, (n = 4; 4500x, 22500x & 34000x) demonstrated myofibrils well-organized into sarcomeres with clear aligned Z-discs.
Fig 6.
Intracellular calcium cycling in hiPSC-CMs.
A) Line-scan images and temporal profiles of Fluo-4 fluorescence of hiPSC-CMs seeded on flat plate or on aligned nanofiber-coated coverslips. Ca+2 transients were evoked by electrical field stimulation at 0.5 Hz. B) Summary graphs showing average data for Ca+2 transient amplitude, time to peak and exponential decay constant (τ) of Ca+2 transients recorded in cells from flat plate (Flat, n = 10) and aligned nanofiber (Nano, n = 14) groups, respectively. *P<0.05 vs standard flat plate group. HiPSC-CMs cultured on aligned nanofibers have faster calcium cycling rate and higher contraction frequency than cells maintained on a flat surface.
Fig 7.
Electrical properties of hiPSC-CMs.
A) Representative action potential (AP) traces recorded during 1 Hz stimulation in patch-clamped single human iPS-CM seeded on flat bottom or on aligned nanofiber-coated coverslips. Dotted lines indicate 0 mV level. B) Summary graphs showing average data for AP amplitude and AP duration at 50 (APD50) and 90 (APD90) % repolarization levels, respectively. Data was recorded in cells from flat plate (Flat, n = 5) and aligned nanofiber (Nano, n = 4) groups, respectively. Resting potential was -74± 2 mV in flat bottom, and -74± 1 mV in aligned nanofiber groups. C) Representative action potential (AP) traces recorded in syncytium of human iPS-CM seeded on flat bottom or on aligned nanofiber-coated coverslips. APs were evoked by electrical field stimulation at 1 Hz and were recorded using voltage-sensitive dye di-4-AN(F)EPPTEA. D) Summary graphs showing average data for AP amplitude and AP duration. Data was obtained both flat plate (Flat, n = 6) and aligned nanofiber (Nano, n = 6) preparations, respectively.
Fig 8.
Assessment of cardiac genes by qRT-PCR.
mRNA levels of three cardiac markers connexin-43 (GJA1), a-actinin (ACTN2), and troponin-I (TNNC1). A) hiPSC-CMs had approximately two fold greater levels of GJA1 mRNA compared to human heart tissue (human 0.78 vs hiPS-CM on flat plates 2.69, n = 6, p = 0.001) B) No significant differences in a-actinin mRNA were found between groups C) hiPSC-CMs of both groups had approximately 1/3 the amount of troponin-I mRNA compared to non-failing human heart tissue (human 1.06 vs hiPSC-CMs on flat plates 0.38, n-6, p = 0.01).