New study identifies protein essential to form stem cell-derived human heart cells

By Bekah McBride

Lab-grown human heart cells provide a powerful tool to understand and potentially treat heart disease. However, the methods to produce human heart cells from pluripotent stem cells are not optimal. Fortunately, a new study out of the University of Wisconsin-Madison Stem Cell & Regenerative Medicine Center, is providing key insight that will aid researchers in growing cardiac cells from stem cells.

The new study on the cover of eLife. Credit: eLife and Jianhua Zhang

The research, which was published June 27 in eLife, investigates the role of extracellular matrix (ECM) proteins in the generation of heart cells derived from human pluripotent stem cells (hPSCs). The ECM fills the space between cells providing structural support and regulating formation of tissues and organs. With a better understanding of ECM and how that impacts heart development, researchers will be able to more effectively develop heart muscle cells, called cardiomyocytes, within the lab that could have potential uses for cardiac repair, regeneration, and cell therapy.

“The better we understand how the soluble factors as well as the ECM proteins work in the cell culture and differentiation, the closer we get to our goals,” said Jianhua Zhang, a senior scientist, who led the study along with Tim Kamp, who serves as director of the Stem Cell and Regenerative Medicine Center and Cellular and Molecular Arrhythmia Research Program.

Zhang shared that researchers have been looking to improve hPSC-cardiomyocytes (hPSC-CMs) differentiation, or the ability to take hPSCs, which can self-renew indefinitely in culture while maintaining the ability to become almost any cell type in the human body, and turn them into heart muscle cells. “However, how the ECM impacts the generation of hPSC-CMs has been largely overlooked,” Zhang said.

To investigate the role of ECM in promoting this cardiac differentiation of hPSCs, Zhang tested a variety of human recombinant and defined ECM proteins to see how they impacted the stem cell growth and differentiation. Specifically, Zhang looked at ECM proteins including laminin-111, laminin-521, fibronectin (FN), and collagen.

Photo of Jianhua
Jianhua Zhang, PhD, Senior Scientist Credit: Clint Thayer

“Our study showed ECM proteins play significant roles in the hPSC adhesion, growth, and cardiac differentiation. And FN plays an essential role and is indispensable in hPSC cardiac differentiation,” said Zhang. “By understanding the roles of ECM, this study will help to develop more robust methods and protocols for generation of hPSC-CMs. Furthermore, this study not only helps in the field for cardiac differentiation, but also other lineages differentiation as well.”

While this study provides important insight into heart cell development, it is built upon a previous study Zhang led which looked at the most efficient way to develop cardiac differentiation of stem cells.

“This study is actually a follow-up paper to the Matrix Sandwich Method that we developed for efficient cardiac differentiation of hPSCs”, Zhang said. The paper, which was published in Circulation Research in 2012, showcased the vital role that ECM plays in the development of hPSC-CMs.

“In order to culture the stem cells, we needed to have an ECM layer on the bottom of the plate. Otherwise, the stem cells would not attach to the plate,” Zhang said. “We would

Matrix Sandwich
Phase contrast and fluorescence images of the matrix sandwich culture of DF19-9-11T iPSCs grown for 4 days and immunolabeled using anti-FN antibody, compared with the monolayer culture. Scale bar is 200 µm. Credit: eLife

then add another layer of ECM on top of the growing stem cells, and we found that this helped promote the most effective differentiation.”

While it was clear that this layering, or sandwich, method more efficiently and reproducibly differentiated hPSC-CMs, researchers did not fully understand why. This latest study explains why the ECM layers were crucial in the differentiation of hPSC-CMs and identifies FN as a key ECM protein in the development of hPSC-CMs.

“The most exciting part of this study is now I understand why the Matrix Sandwich Method worked. We were able to identify the FN and its integrin receptors as well as the downstream signaling pathways in this study,” Zhang explained. “With a better understanding ECM’s roles in stem cell growth and cardiac differentiation we now hope to investigate the roles of FN and other ECM proteins in promoting the hPSC-CMs transplantation for cell therapy.”

Zhang shared that this next step could help researchers to realize the full potential of using hPSC-CMs for disease modeling, drug screening, cardiac regeneration, and cell therapy. This is very meaningful to Zhang who began working in cardiovascular research more than 16 years ago and has published more than 25 peer-reviewed, high impact papers.

“I became interested in stem cell and heart research when I began working with the stem cells and saw them turning into heart cells beating in a cell culture dish under a microscope,” Zhang said. “It was amazing. I’ve become more and more dedicated to this research, and I can really see the potential of using the stem cell technologies to cure disease and improve our health.”