Generation of NKX2.5^GFP Reporter Human iPSCs and Differentiation Into Functional Cardiac Fibroblasts

Area of Science:

• Cardiovascular Biology
• Stem Cell Biology
• Regenerative Medicine

Background:

• Direct cardiac reprogramming offers potential for heart regeneration by converting fibroblasts to cardiomyocytes.
• Current human cell studies are limited by a lack of reporter fibroblasts, hindering factor screening and protocol development.

Purpose of the Study:

• To generate functional human NKX2.5^GFP reporter cardiac fibroblasts.
• To establish a reliable cellular model for advancing direct cardiac reprogramming research.

Main Methods:

• Generated a human induced pluripotent stem cell (hiPSC) line with a CRISPR-Cas9 knock-in NKX2.5^GFP reporter.
• Directed differentiation of NKX2.5^GFP hiPSCs to obtain reporter cardiac fibroblasts.
• Validated reporter fidelity and fibroblast functionality.

Main Results:

• The NKX2.5^GFP reporter accurately tracked NKX2.5 expression in differentiated hiPSCs and their cardiac progeny.
• Generated NKX2.5^GFP cardiac fibroblasts exhibited typical morphology, marker expression, and function.
• The reporter system allows for the detection of both cardiomyocytes and cardiovascular progenitors.

Conclusions:

• The developed NKX2.5^GFP reporter fibroblasts provide an unlimited cellular model supply for cardiac reprogramming research.
• These reporter lines are valuable tools for human direct cardiac reprogramming studies.
• This advancement facilitates progress in developing effective cardiac regeneration strategies.