Kyle Loh, PhD
Developmental Biology
Patients with single ventricle (SV) heart defects often develop long-term circulatory complications after undergoing several open-heart surgeries to reroute the blood network. What remains unknown is which blood vessels in the circulatory system - the arteries, veins, and lymphatics - influence the abnormalities associated with SV disease.
"It's important to differentiate each of these major blood vessels,” says Kyle Loh, PhD, Assistant Professor in the Department of Developmental Biology at Stanford School of Medicine. Each of these plays a specific role in the blood circulation to the heart. "Understanding the differences in each of these blood vessels would let us know, for instance, that the lymphatic system is sick, but the arteries and veins aren't," he explains.
Dr. Loh is leading a project with co-investigator Marlene Rabinovitch, MD, Professor of Pediatric Cardiology, to understand lymphatic, arterial, and venous dysfunction in SV disease using stem cells.
With the support of $300,000 from MCHRI through the Additional Ventures Innovation Fund Single Ventricle Disease Research Awards Program, Dr. Loh is conducting this initiative to understand what causes the SV disease, and in particular, if there are defects in any of the blood vessels.
"We have to understand how the blood vessels are normally built in the early development to understand how those blood vessels go awry or improperly formed in a congenital defect, like a single ventricle disease," says Dr. Loh.
Understanding blood vessels in the early development
The project will use human stem cells collected from healthy individuals and SV patients and be differentiated into arterial, venous, and lymphatic cells. Through this approach, Dr. Loh and his team aim to learn how blood vessels take shape and form and how they may account for defects in SV heart disease, resulting in vascular complications.
Because human stem cells can make thousands of different cell types, it's challenging to direct stem cells into a specific, differentiated body cell, says. Dr. Loh. It's like getting into your car, and there are multiple ways to get to your destination. How do you force the stem cells only to go down a single path?
That's how Dr. Loh and his colleagues spent the first six months of their project establishing a robust procedure for differentiating artery and venous cells. In the early stage of development, stem cells face choices in what they become. They can become a specific kind of tissue cell, like an artery or vein. Or they can become a different kind of cell.
Akin to driving and pulling up at T-intersection, there would be a green light signal for a turning left and a detour sign for the right side, blocking the car from going in that direction. Using this concept and understanding this process at each juncture, Dr. Loh and his team signaled cells to become cell type A while inhibiting for other cells to make cell type B.
"Using this strategic approach, we were activating the signals we wanted and inhibiting the ones we didn't want," says Dr. Loh. "We forced the stem cells to go down on the desired path."
They then used single-cell RNA sequencing to profile the artery cells and learned they made over 90% pure artery cells. Meaning, they were able to generate a 90% high purity in their cell differentiation where at least nine out of ten cells in the Petri dish were of the desired type, such as artery cells (instead of an impure mix of multiple cell types).
"We want to use this approach of guiding new healthy human stem cells into the arteries and veins and apply it to single ventricle disease stem cells to get them to become arteries and veins," says Dr. Loh. While they may not be the same ones as the healthy ones, that's where single-cell RNA sequencing will help understand the differences in each cell.
Moving forward, the team aims to use the same approach using stem cells obtained from SV patients, turn them into artery and vein tissue, and compare them to the healthy ones. Ultimately, the goal is to understand this process, enabling targeted therapeutic interventions for SV disease.
"This knowledge would help us precisely target therapies to fix the lymphatics or other specific areas," says Dr. Loh.
The project is currently in progress. This story is a complimentary piece to a larger article. To read the article, click here.
BY ROXANNA VAN NORMAN
Roxanna Van Norman is the marketing manager for the Stanford Maternal and Child Health Research Institute.