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Enhancing the Engraftment of Human Induced Pluripotent Stem Cell-derived Cardiomyocytes via a Transient Inhibition of Rho Kinase Activity
Published on: July 10, 2019
Rock inhibitor may compromise human induced pluripotent stem cells for cardiac differentiation in 3D
Bin Jiang1, Wenquan Ou1, James G Shamul1
1Fischell Department of Bioengineering, University of Maryland, College Park, MD, 20742, United States.
Insights
Optimizing Rock inhibitor (RI) concentration is key for homogeneous 3D cardiac differentiation of human induced pluripotent stem cells (iPSCs). Lowering RI improves cardiomyocyte yield and functional maturity for heart disease research and drug screening.
Area of Science:
- Stem Cell Biology
- Cardiovascular Research
- Biomedical Engineering
Background:
- Human induced pluripotent stem cells (iPSCs) are crucial for studying heart diseases and drug development.
- Achieving homogeneous 3D cardiac differentiation from iPSCs remains a significant challenge.
- Standard Rock inhibitor (RI) concentrations can induce undesirable germ layer differentiation, compromising cardiac differentiation quality.
Purpose of the Study:
- To investigate the effect of Rock inhibitor (RI) concentration on 3D cardiac differentiation of human iPSCs.
- To optimize RI dosage for enhanced homogeneity and efficiency in cardiac differentiation.
- To develop a robust platform for generating high-quality, functional cardiomyocytes from iPSCs.
Main Methods:
- Human iPSCs were cultured in 3D conditions with varying RI concentrations (10 μM vs. 1 μM).
- Cardiac differentiation efficiency and homogeneity were assessed by beating efficiency and onset beating time (OBT).
- Resultant cardiomyocytes were characterized for ultrastructure and drug responsiveness, and cultured in GelMA hydrogels.
Main Results:
- Reducing RI to 1 μM significantly improved iPSC pluripotency and spheroid quality compared to 10 μM.
- Beating efficiency of 3D cardiac differentiation increased to over 95% within 7 days with 1 μM RI.
- Synchronized OBT within 1 day and formation of synchronously beating 3D constructs demonstrated high functional homogeneity.
Conclusions:
- Optimized RI concentration (1 μM) enables highly efficient and homogeneous 3D cardiac differentiation of human iPSCs.
- This optimized method yields high-quality cardiomyocytes with mature functional characteristics.
- The findings facilitate the use of iPSC-derived cardiomyocytes for heart disease research and therapeutic applications.
Abstract:
Cardiomyocytes differentiated from human induced pluripotent stem cells (iPSCs) are valuable for the understanding/treatment of the deadly heart diseases and their drug screening. However, the very much needed homogeneous 3D cardiac differentiation of human iPSCs is still challenging. Here, it is discovered surprisingly that Rock inhibitor (RI), used ubiquitously to improve the survival/yield of human iPSCs, induces early gastrulation-like change to human iPSCs in 3D culture and may cause their heterogeneous differentiation into all the three germ layers (i.e., ectoderm, mesoderm, and endoderm) at the commonly used concentration (10 μM). This greatly compromises the capacity of human iPSCs for homogeneous 3D cardiac differentiation. By reducing the RI to 1 μM for 3D culture, the human iPSCs retain high pluripotency/quality in inner cell mass-like solid 3D spheroids. Consequently, the beating efficiency of 3D cardiac differentiation can be improved to more than 95 % in ~7 days (compared to less than ~50 % in 14 days for the 10 μM RI condition). Furthermore, the outset beating time (OBT) of all resultant cardiac spheroids (CSs) is synchronized within only 1 day and they form a synchronously beating 3D construct after 5-day culture in gelatin methacrylol (GelMA) hydrogel, showing high homogeneity (in terms of the OBT) in functional maturity of the CSs. Moreover, the resultant cardiomyocytes are of high quality with key functional ultrastructures and highly responsive to cardiac drugs. These discoveries may greatly facilitate the utilization of human iPSCs for understanding and treating heart diseases.
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