Optically-driven maturation of human pluripotent stem cell-derived cardiomyocytes via a far-red sensitive organic semiconductor

Technical Summary: Optically-driven maturation of human pluripotent stem cell-derived cardiomyocytes via a far-red sensitive organic semiconductor

Problem Statement
Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) are critical resources for preclinical research, personalized medicine, and drug testing. However, a major translational bottleneck is their incomplete maturation; hPSC-CMs typically retain fetal-like morphological, metabolic, and functional characteristics that limit their utility in modeling late-onset diseases and therapeutic applications. While various strategies exist to enhance maturation—including long-term culturing, metabolic media optimization, 3D tissue engineering, and electrical or mechanical stimulation—these approaches often suffer from limitations regarding effectiveness, technical complexity, cost, or the necessity for genetic manipulation. Furthermore, existing optogenetic tools using green-absorbing polymers (e.g., P3HT) are limited by poor tissue penetration. Previous studies using far-red absorbing polymers have shown mixed results, with some reporting cell damage or attributing effects to substrate nanostructure rather than photoexcitation.

Methodology
The study investigates the effects of chronic (long-term) photostimulation on hPSC-CMs using a far-red absorbing conjugated polymer, PCPDTBT (poly[2,1,3-benzothiadiazole-4,7-diyl[4,4-bis(2-ethylhexyl)−4H-cyclopenta[2,1-b:3,4-b']dithiophene-2,6-diyl]]).

• Experimental Setup: hPSC-CMs were seeded on thin films of PCPDTBT coated with fibronectin and laminin. Control groups included cells on glass substrates and cells on PCPDTBT kept in the dark.
• Stimulation Protocol: Cells on PCPDTBT were subjected to a "chronic" optical stimulation protocol for 6 hours. This involved pulsed red light (peak emission 660 ± 20 nm) with a duty cycle of 400 ms light followed by 4000 ms darkness, at a power density of 0.6 mW/mm².
• Multi-level Analysis: The study employed a comprehensive suite of assays to assess maturation:
  • Transcriptional: Bulk RNA sequencing (RNA-seq) and Gene Set Enrichment Analysis (GSEA) to identify pathway modulation.
  • Electrophysiological: Patch-clamp recordings to measure maximal diastolic potential (MDP), action potential (AP) parameters, and net membrane currents (NMCs) via voltage ramps.
  • Calcium Dynamics: Measurement of Ca²⁺ transient amplitude and decay kinetics using FLUO4AM, with SERCA inhibition tests using thapsigargin.
  • Morphological: Immunofluorescence staining for α-sarcomeric actinin and Bin-1 (a T-tubule marker), coupled with image analysis for cell area, roundness, and T-tubule organization.
  • Metabolic: Scanning Electrochemical Microscopy (SECM) using microbiosensors to quantify glucose uptake, lactate release, and oxygen consumption at the single-cell level, including experiments with Albumax to test fatty acid oxidation.

Key Contributions and Results
The study demonstrates that 6-hour pulsed photostimulation via PCPDTBT significantly enhances hPSC-CM maturation across multiple biological dimensions without inducing phototoxicity.

1. Transcriptional Remodeling: RNA-seq revealed that PCPDTBT photostimulation induces a specific transcriptional program linked to maturation. Key findings include:

   • Metabolic Shift: Upregulation of genes involved in fatty acid metabolism (e.g., ME1) and oxidative phosphorylation, and downregulation of glycolysis-associated genes (e.g., LDHA).
   • Electrical Maturation: Upregulation of ion channel genes (SCN5A, KCNJ3) and pathways related to membrane polarization and calcium signaling.
   • Safety: No significant upregulation of apoptotic or pro-inflammatory pathways was observed.

2. Electrophysiological Improvements:

   • Cells exposed to chronic light stimulation exhibited a significantly hyperpolarized Maximal Diastolic Potential (MDP), reaching values (~-75.5 mV) closer to adult cardiomyocytes (-80 to -85 mV) compared to controls.
   • There was a higher proportion of quiescent (non-spontaneously beating) cells, a hallmark of mature cardiomyocytes.
   • Net membrane current analysis indicated a shift toward increased outward conductances and reduced inward currents, consistent with the hyperpolarization.

3. Enhanced Calcium Handling:

   • Photostimulation increased Ca²⁺ transient amplitude and accelerated Ca²⁺ decay kinetics (faster half-time decay).
   • The accelerated decay was reversed by SERCA inhibition, suggesting that PCPDTBT photostimulation enhances SERCA-dependent Ca²⁺ reuptake, indicative of improved excitation-contraction coupling.

4. Structural Maturation:

   • Cells showed a significant increase in cell area and a decrease in roundness, adopting a more elongated, rod-like shape.
   • Immunofluorescence revealed organized T-tubule structures, evidenced by the colocalization of Bin-1 and α-sarcomeric actinin, a feature typically absent in immature hPSC-CMs.

5. Metabolic Switch:

   • SECM measurements confirmed a functional metabolic shift: photostimulated cells exhibited decreased glucose uptake and lactate release, alongside increased oxygen consumption.
   • The presence of Albumax (lipid source) further enhanced oxygen consumption in stimulated cells, confirming a transition from glycolysis to fatty acid β-oxidation.

Significance and Claims
The authors claim that this work establishes a simple, cost-effective, and non-invasive strategy to promote the morpho-functional and metabolic maturation of hPSC-CMs. By leveraging the biophysical properties of a far-red sensitive organic semiconductor, the approach offers a "gene-free" tool with high spatial and temporal precision.

The study positions this method as a practical alternative to complex 3D culture systems or genetic editing. It highlights that the specific choice of photoactive material (PCPDTBT) and the optimization of the stimulation protocol (pulsed, low-intensity, long-duration) are crucial for achieving maturation without the cytotoxicity observed in previous studies with other polymers. The authors assert that this approach expands the applicability of hPSC-CMs for in vitro disease modeling, drug testing, and future therapeutic development by providing a more adult-like cellular phenotype. The work contributes to the emerging field of biohybrid regenerative bioelectronics by demonstrating light as a "touchless" trigger for cellular maturation.