Development of Novel Strategies for Enriching Functionally Mature Cardiomyocytes Derived from Human Pluripotent Stem Cells

The capacity of cardiomyocytes differentiated from human pluripotent stem cells (hPSC-CMs) provides an unlimited resource for human CMs for a wide range of applications such as cell based cardiac repair, cardiac drug toxicology screening, and human cardiac disease modeling. However, applicability is significantly limited by their immature phenotypes. It has been well known that currently available hPSC-CMs represent immature embryonic or fetal stage CMs and are functionally and structurally different from mature human primary CMs.To overcome this critical issue, several new approaches aiming to generate more mature hPSC-CM have been developed. However, methodologies for maturing hPSC-CMs are rapidly improving; even these improved methods only result in heterogeneous populations of hPSC-CMs of varying maturities. These cell populations include a significant amount of immature hPSC-CMs which may elicit inaccurate outcomes in many application using hPSC-CMs. Thus, it is necessary to develop a method that can selectively purify mature hPSC-CMs. As another limitation, currently available methodologies for maturing hPSC-CMs are small scale. While these methods are useful for small-scale research, they are not scalable and difficult to use for large scale research or clinical application.Accordingly, the goal of the current proposal are: 1) to develop a novel system which can purify homogeneous populations of mature hPSC-CMs via antibodies (Abs) targeting surface protein markers that are uniquely expressed in mature CMs. 2) In addition, we will develop new protocols for inducing hPSC-CM maturation on a large scale based on the use of several postnatal hormones.To achieve these aims, we have identified a list of candidate surface proteins with protein expressions that are specifically limited to primary neonatal (1 day old) CMs from our preliminary proteomic studies. Among the candidate surface proteins, throughout the series of preliminary experiments, we have selected and hypothesized that the CD36 surface protein and CD36 antibody could be used to distinguish functionally mature hPSC-CMs. By using CD36 Ab, we will first enrich the CD36-positive hPSC-CMs (CD36+hPSC-CMs) from the hPSC-CM cultures that will be generated by ourin vitroCM maturation systems. Subsequently, these enriched CD36+hPSC-CMs will be examined in terms of CM identity and functional maturity using various structural, functional and physiological assays.We anticipate that this study will provide the first evidence for identifying the novel surface markers that can distinguish mature CMs at neonatal stage from a pool of immature CMs. We will also develop a novel protocol for large scale hPSC-CM maturation.