Evaluation of Changes in Morphology and Function of Human Induced Pluripotent Stem Cell Derived Cardiomyocytes (HiPSC-CMs) Cultured on an Aligned-Nanofiber Cardiac Patch

<div><p>Introduction</p><p>Dilated cardiomyopathy is a major cause of progressive heart failure. Utilization of stem cell therapy offers a potential means of regenerating viable cardiac tissue. However, a major obstacle to stem cell therapy is the delivery and survival of implanted stem cells in the ischemic heart. To address this issue, we have developed a biomimetic aligned nanofibrous cardiac patch and characterized the alignment and function of human inducible pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) cultured on this cardiac patch. This hiPSC-CMs seeded patch was compared with hiPSC-CMs cultured on standard flat cell culture plates.</p><p>Methods</p><p>hiPSC-CMs were cultured on; 1) a highly aligned polylactide-co-glycolide (PLGA) nanofiber scaffold (~50 microns thick) and 2) on a standard flat culture plate. Scanning electron microscopy (SEM) was used to determine alignment of PLGA nanofibers and orientation of the cells on the respective surfaces. Analysis of gap junctions (Connexin-43) was performed by confocal imaging in both the groups. Calcium cycling and patch-clamp technique were performed to measure calcium transients and electrical coupling properties of cardiomyocytes.</p><p>Results</p><p>SEM demonstrated >90% alignment of the nanofibers in the patch which is similar to the extracellular matrix of decellularized rat myocardium. Confocal imaging of the cardiomyocytes demonstrated symmetrical alignment in the same direction on the aligned nanofiber patch in sharp contrast to the random appearance of cardiomyocytes cultured on a tissue culture plate. The hiPSC-CMs cultured on aligned nanofiber cardiac patches showed more efficient calcium cycling compared with cells cultured on standard flat surface culture plates. Quantification of mRNA with qRT-PCR confirmed that these cardiomyocytes expressed α-actinin, troponin-T and connexin-43 <i>in-vitro</i>.</p><p>Conclusions</p><p>Overall, our results demonstrated changes in morphology and function of human induced pluripotent derived cardiomyocytes cultured in an anisotropic environment created by an aligned nanofiber patch. In this environment, these cells better approximate normal cardiac tissue compared with cells cultured on flat surface and can serve as the basis for bioengineering of an implantable cardiac patch.</p></div

Electrical properties of hiPSC-CMs.

<p><b>A)</b> 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>B)</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. <b>C)</b> 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. <b>D)</b> 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.</p

Analysis of gap junctions in hiPSC-CMs by confocal microscopy in cells seeded on flat plate and aligned nanofiber.

<p>The gap junctions in hiPSC-CMs was evaluated by CX-43 immunostaining of human cardiomyocytes seeded on flat surface versus aligned-nanofiber coated coverslips. <b>A</b>) Flat plate, CX-43 (Red), SAA (Green) and DAPI (Blue); <b>B</b>) Aligned nanofiber, CX-43 (Red), SAA (Green) and DAPI (Blue); <b>C</b>) 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).</p

Primer list.

<p>Primer list.</p

SEM imaging of Human iPSC-Cardiomyocytes.

<p>Cells seeded on <b>A)</b> Flat surface vs <b>B)</b> Aligned-nanofiber coated coverslips (600x & 1200x) show different cell morphology.</p

Intracellular calcium cycling in hiPSC-CMs.

<p><b>A)</b> 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>B)</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.</p

Assessment of cardiac genes by qRT-PCR.

<p>mRNA levels of three cardiac markers connexin-43 (GJA1), a-actinin (ACTN2), and troponin-I (TNNC1). <b>A)</b> 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>B)</b> No significant differences in a-actinin mRNA were found between groups <b>C)</b> 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).</p

Confocal Imaging of mitochondria.

<p>Mito-tracker red staining shows alignment of Human iPSC-Cardiomyocytes seeded on <b>A)</b> Flat surface vs <b>B)</b> Aligned-nanofiber coated coverslips (32x & 200x). <b>C)</b> TEM imaging showing comparison of mitochondrial morphology and arrangement of hiPSC-CMs seeded on flat plate versus aligned nanofiber groups.</p

Mechanical properties for aligned PLGA nanofiber scaffolds.

<p>Ultimate tensile strength (UTS) and Young’s modulus are recorded in megaPascals (MPa) with the standard deviation (SD) shown.</p><p>Mechanical properties for aligned PLGA nanofiber scaffolds.</p

The alignment of hiPSC-CMs.

<p>Cells seeded on <b>A)</b> Flat surface plate and <b>B)</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.</p