AVC-Like Cardiomyocyte Induction for Slower Impulse Propagation
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Solution Overview
Problem
Current methods for generating atrioventricular canal (AVC) cardiomyocytes from human pluripotent stem cells (hPSCs) are lacking in robustness, complexity, and reproducibility, and the generated cells do not exhibit key characteristics such as slower impulse propagation.
Innovation Solution
A method involving incubation of cardiogenic mesoderm cells with WNT2 and retinoic acid (RA) to generate AVC-like cells that express TBX3 and at least one of BMP2, MSX2, TBX2, optionally with MESP1/2 and EOMES, and potentially including BMP ligands like BMP4, to achieve desired electrophysiological properties.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If BMP4 ligand is used to generate AVC-like cells from mesoderm, then AVC-like cell generation is achieved, but the cells do not display slower impulse propagation and the method has complexity and reproducibility issues
Solution Approach 1:
The patent changes the chemical parameters of the induction medium by replacing BMP4 with a defined combination of WNT3a (10-50 ng/mL), IWP2 (100-500 nM), and RA (100-500 nM). This parameter modification resolves the contradiction by achieving reliable AVC-like cell generation (improving reliability) while using a defined medium that enhances reproducibility and reduces method complexity.
Solution Approach 2:
The patent introduces WNT3a as an intermediary substance that mediates the differentiation process. By using WNT3a in combination with IWP2 and RA, the method achieves reliable AVC-like cell generation without the complexity and reproducibility issues associated with BMP4-based approaches.
2Manufacturing precision
If BMP4 ligand is used to generate AVC-like cells, then cell generation is achieved, but the generated cells lack key electrophysiological characteristics
Solution Approach 1:
The patent modifies the chemical parameters of the induction medium by using WNT3a (10-50 ng/mL), IWP2 (100-500 nM), and RA (100-500 nM) instead of BMP4. This parameter change ensures that the generated cells display authentic AVC electrophysiological characteristics including slower impulse propagation, while maintaining reliable cell generation.
3Productivity
If complex protocols are used for AVC cardiomyocyte generation, then cell generation is achieved, but reproducibility is poor
Solution Approach 1:
The patent employs a defined medium with specific concentrations of WNT3a (10-50 ng/mL), IWP2 (100-500 nM), and RA (100-500 nM). This precise parameter specification enables both efficient AVC-like cell generation and high reproducibility across different experiments and laboratories.
Solution Approach 2:
The patent uses a defined medium formulation that can be easily prepared and standardized, replacing complex undefined serum-based systems. This approach improves reproducibility while maintaining efficient cell generation.
Data Source
Figure 1A~1H
Figure 2
Figure 3A~3B
AI summary
The present disclosure provides a method of generating AVC-like cell(s) or AVC-like cardiomyocyte(s) from cardiogenic mesoderm cells comprising incubating the cardiogenic mesoderm cells in an AVC cardiomyocyte induction medium comprising WNT2 and retinoic acid (RA) to obtain the AVC-like cell(s). Preferably, the AVC-like cell(s) express TBX3 and at least one of BMP2, MSX2 and TBX2.