Geometric Control of Cardiomyogenic Induction in Human Pluripotent Stem Cells

Abstract

Pluripotent stem cells provide the opportunity to study human cardiogenesis in vitro, and are a renewable source of tissue for drug testing and disease models, including replacement cardiomyocytes that may be a useful treatment for heart failure. Typically, differentiation is initiated by forming spherical cell aggregates wherein an extraembryonic endoderm (ExE) layer develops on the surface. Given that interactions between endoderm and mesoderm influence embryonic cardiogenesis, we examined the impact of human embryonic stem cell (hESC) aggregate size on endoderm and cardiac development. We first demonstrated aggregate size control by micropatterning hESC colonies at defined diameters and transferring the colonies to suspension. The ratio of endoderm (GATA-6) to neural (PAX6) gene and protein expression increased with decreasing colony size. Subsequently, maximum mesoderm and cardiac induction occurred in larger aggregates when initiated with endoderm-biased hESCs (high GATA-6:PAX6), and in smaller aggregates when initiated with neural-biased hESCs (low GATA-6:PAX6). Additionally, incorporating micropatterned aggregates in a stirred suspension bioreactor increased cell yields and contracting aggregate frequency. We next interrogated the relationship between aggregate size and endoderm and cardiac differentiation efficiency in sizecontrolled aggregates, generated using forced aggregation, in defined cardiogenic medium.

Design and formulation of functional pluripotent stem cell-derived cardiac microtissues

“…Cardiac differentiation of human embryonic stem cells was carried out as reported previously (44). In this study, the HES2 (ES Cell International) hESC line was used.…”

Production of Large Numbers of Size-controlled Tumor Spheroids Using Microwell Plates

“…In addition to investigating the mechanical and morphometric properties of spheroids, assembly in the microwell system permits large-scale production of mixed-composition spheroids, consisting of combinations of multiple cell types and / or biomaterials 6,7 . The interactions of tumor cells with other cell types are important to more closely model the behavior of tumors in vivo 14 , thus it may be of interest to generate spheroids from tumor cells in combination with fibroblasts and endothelial cells, for example.…”

“…Employing forced aggregation within a series of densely packed micron-scale wells (see Figure 1), this approach permits the generation of spheroids from arbitrary numbers of cells, including mixtures of multiple cell types 6 , as well as the incorporation of various biomaterials 7 . Spheroids are formed in large numbersthousands to hundreds of thousands or more -and may be extracted immediately or maintained in the microwells in which they were formed with medium exchange for at least one 8 to two (unpublished observation) weeks.…”

Chemically controlled aggregation of pluripotent stem cells

“…Aggregate size control has emerged as a critical parameter in understanding and directing cell fate transitions, as cells sense and respond to cues in their environment that are influenced by geometry both in vitro and in vivo. When recapitulating developmental processes in vitro to study cell biology and create cell therapies, controlling aggregate size is essential for the homogeneity, reproducibility, and efficiency of the desired protocol (Bauwens et al, 2011). …”