(262e) Effects of Tunable Experimental Parameters on Structural Maturity of hiPSC Derived Cardiomyocytes

Cardiovascular disease is the leading cause of death worldwide (Ahmed et al., 2020). To effectively treat cardiovascular disease, cardiomyocytes (CMs), the contractile heart cells, derived from human-induced pluripotent stem cells can be used for disease modeling and regeneration therapies. These CMs can be produced in a 3D environment from hiPSCs encapsulated in a hydrogel (Finklea et al., 2021). The hiPSCs are encapsulated in a hydrogel (PEG-Fibrinogen) using a custom microfluidic device (Tian & Lipke, 2020), that controls the initial shape and size of the hiPSC-hydrogel structure. These hiPSC-hydrogel structures are called microspheroids, which are spherical or rod-shaped. The hiPSCs encapsulated in these spheroids are shown to provide consistent CM efficiency upon differentiation (Finklea et al., 2021).
The microfluidic device produces microspheroids of different sizes and shapes. Our hypothesis is that by tuning the key structural features (axial ratio and diameter) of the microfluidic device, and the cell concentration in the microspheroids, CMs with different structural features are obtained upon differentiation. The axial ratio of the microspheroid denotes the ratio of the axial diameter to the radial diameter of the microspheroids, and the diameter of the microspheroid denotes the radial diameter. Cell features such as cell area, circularity, eccentricity, elongation, and sarcomere properties such as length, orientation index, and organization score were measured from the cell samples on differentiation day 30.
Based on the literature, mature cells have sarcomere lengths of 2.2 μm (Campostrini et al., 2021). They tend to be more elongated, leading to higher eccentricity and elongation. The cell area of mature CMs is at least 5000 μm^2 (Vu & Kofidis, 2014). Despite having these descriptions of mature CMs, there is a lack of reported feature ranges that define structural maturity. Identifying the feature ranges that define structural maturity is essential to find mature CMs. Subsequently, the identified mature CMs can be related to the initial experimental parameters to find the relationship between the experimental parameters and the structural maturity.
In this research, maturity index (m-index) is defined to identify potential structurally mature CMs based on the measured cell features. The m-index is calculated as follows. The cell features were individually assigned a rank between 1 and 5 for each cell. Since structurally mature cells are elongated, they exhibit high values in the cell area, eccentricity, elongation, sarcomere orientation index, and organization score, and low values in circularity. For all features except circularity, high values suggest greater structural maturity. Conversely, lower values of circularity indicate high maturity. Each feature is divided into five percentiles (0-20th,20-40th,40-60th,60-80th, 80-100th). Features other than circularity are categorized such that the values above the 80th percentile were ranked as 5, signifying high maturity, while those below the 20th percentile were ranked 1, indicating low maturity. Ranks 2, 3, and 4 consist of cells with values falling between the 20-40th, 40-60th, and 60-80th percentiles, respectively. Regarding circularity, the values below the 20th percentile were ranked as 5, reflecting the low values of circularity with high maturity, and values above the 80th percentile are ranked as 5, indicating low structural maturity. Conversely, regarding circularity, a rank of 1 represents high maturity, while 5 indicates low maturity, because low value of circularity indicates high maturity. The individual feature ranks are then averaged to find the m-index. An arbitrary cut-off value of 3.5 was chosen to define the potential structurally mature CMs. Figure 1 shows the potential structurally mature and immature CMs classified by the m-index. The potential structurally mature CMs have high cell area, eccentricity, elongation, sarcomere length, sarcomere organization score, sarcomere orientation index. The two feature ranges overlap for almost all the features, and there were not any distinct features individually representing maturity based on m-index. The plots for elongation vs other features show that all cells are potentially mature when elongation is greater than 6. However, this value is subject to change when the arbitrary cut-off value for the m-index is changed.
The percentage of potential structurally mature CMs from each batch was calculated, and the Pearson (Cohen et al., 2009) and Spearman correlation (Myers & Sirois, 2004) between the percentage of mature CMs and the tunable experimental parameters were evaluated. Among the three tunable experimental parameters, the axial ratio has the highest Pearson correlation (r=0.33) and high Spearman correlation (ρ=0.4) with the percentage of mature CMs among the three experimental parameters, as shown in Figure 2. The future work in this research involves calculating weighted average m-index to account for the pre-defined feature ranges of cell area, and sarcomere length. Also, a robust m-index cut-off to define structurally mature CMs will be developed by analyzing different cut-off values with the potential relationship the corresponding mature CMs form with the tunable parameters. A regression model will be built to identify the potential relationship between the tunable experimental parameters and the cell structural maturity.

References:

Ahmed, R. E., Anzai, T., Chanthra, N., & Uosaki, H. (2020). A Brief Review of Current Maturation Methods for Human Induced Pluripotent Stem Cells-Derived Cardiomyocytes. Front Cell Dev Biol, 8, 178. https://doi.org/10.3389/fcell.2020.00178
Campostrini, G., Windt, L. M., van Meer, B. J., Bellin, M., & Mummery, C. L. (2021). Cardiac tissues from stem cells: new routes to maturation and cardiac regeneration. Circulation Research, 128(6), 775-801.
Cohen, I., Huang, Y., Chen, J., Benesty, J., Benesty, J., Chen, J., Huang, Y., & Cohen, I. (2009). Pearson correlation coefficient. Noise reduction in speech processing, 1-4.
Finklea, F. B., Tian, Y., Kerscher, P., Seeto, W. J., Ellis, M. E., & Lipke, E. A. (2021). Engineered cardiac tissue microsphere production through direct differentiation of hydrogel-encapsulated human pluripotent stem cells. Biomaterials, 274, 120818. https://doi.org/10.1016/j.biomaterials.2021.120818
Myers, L., & Sirois, M. J. (2004). Spearman correlation coefficients, differences between. Encyclopedia of statistical sciences, 12.
Tian, Y., & Lipke, E. A. (2020). Microfluidic Production of Cell-Laden Microspheroidal Hydrogels with Different Geometric Shapes. ACS Biomater Sci Eng, 6(11), 6435-6444. https://doi.org/10.1021/acsbiomaterials.0c00980
Vu, T., & Kofidis, T. (2014). Biomaterials and cells for cardiac tissue engineering. In Cardiac regeneration and repair (pp. 127-179). Elsevier.