Systematic Co-Culture of IPSC-CM and IPSC-AN Promote Co-Maturation In Vitro

<p>The cardiac microenvironment is a complex system of multicellular interactions that enables proper heart function. In native heart muscle, sympathetic neurons (SN) and parasympathetic neurons (PSN) modulate the beat rate of cardiomyocytes (CM) to maintain homeostasis. Pluripotent stem cells can differentiate into CM, but differentiated CMs are fetal-like, with a high beat rate, and lack organized sarcomeric structure. Recent reports of co-cultured pluripotent CM/SN pairs have reported electrophysiological changes in the SN (increased upstroke velocity). However, interpretation of these results are obscured by the differences in co-culture parameters between studies (stage of development, co-culture duration, etc.). The goal of this work was to improve autonomic neuron differentiation efficiency (iPSC-ANs), establish a platform for CM/AN co-culture, and investigate the influence of these parameters on CM/AN crosstalk/responses.</p> <p>A protocol was developed in-house that incorporated <em>in vivo</em> developmental cues. The differentiated progeny were characterized during/after optimizing initial seeding density, RA concentration, and BMP4 concentration. The resulting D19 iPSC-AN population was quantified with Stardist (FIJI plugin) as 50+% NF+, 50+% Phox2B+, and 40% Sox10+. Differentiated autonomic neurons were co-cultured with cardiomyocytes to investigate CM/AN morphologic changes. The selected media composition and CM/AN ratio maintained viable neurocardiac co-cultures. AN projections interacted with CMs, and exhibited target specificity<em> in vitro</em>. CMs continued to contract during co-culture reorganization and was recorded for analysis. There was a significant decrease in the beat rate of co-cultured CMs (p value < 0.001)<em> in all CM+AN co-culture </em>experiments. After completing a systematic analysis of co-culture experiments for contraction strength, surprisingly <em>early </em>CMs co-cultured with <em>early </em>ANs contracted significantly stronger (p value < 0.05, <em>n</em> = 32) than <em>late </em>CMs co-cultured with <em>late </em>ANs.</p> <p>The field of cardiac co-culture research is growing, and a systematic analysis pipeline streamlines observed relationships and conclusions about CM/AN crosstalk. This body of work entails a reliable protocol for iPSC-AN derivation and neurocardiac<em> in vitro</em> modeling for multiple CM/AN combinations. I have provided fundamental understanding of CM behavior in heterotypic models along with insight on AN behavior. CM/AN behavior serves as a building block for more complex CM multicellular models. Consistent CM contraction rate in the presence of ANs suggests that a systematic approach to co-culture could further improve cardiomyocyte Engineered Heart Tissue (EHT) research.</p>

Name thesis:degree_name

Doctor of Philosophy (PhD)

Level thesis:degree_level

Dissertation

Discipline thesis:degree_discipline

Biomedical Engineering

Year

2025

Author dc:creator

• Terrell, William Gregory, Jr.

Contributors dc:contributor

• Tracy Hookway
• Sha Jin
• Ying Wang

• Autonomic neuron
• Cardiomyocytes
• Co-culture
• Heterotypic models
• Innervation
• Stem cell
• Biomedical Engineering and Bioengineering

Repository record dc:identifier

https://orb.binghamton.edu/dissertation_and_theses/439

OAI identifier oai:identifier

oai:orb.binghamton.edu:dissertation_and_theses-1445