Assessing Cardiomyocyte Subtypes Following Transcription Factor-mediated Reprogramming of Mouse Embryonic Fibroblasts

Antonio Fernandez-Perez; Nikhil V. Munshi

doi:10.3791/55456

Assessing Cardiomyocyte Subtypes Following Transcription Factor-mediated Reprogramming of Mouse E...

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Developmental Biology

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JoVE Journal Developmental Biology

Assessing Cardiomyocyte Subtypes Following Transcription Factor-mediated Reprogramming of Mouse Embryonic Fibroblasts

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09:29 min

March 22, 2017

DOI: 10.3791/55456-v

Antonio Fernandez-Perez¹, Nikhil V. Munshi¹

¹Department of Internal Medicine- Cardiology,UT Southwestern Medical Center

Overview

This manuscript describes a step-by-step protocol for generating and quantifying diverse reprogrammed cardiac subtypes. This method addresses key questions regarding subtype-specific lineage conversion during cardiac reprogramming.

Key Study Components

Area of Science

Cardiac reprogramming
Cellular biology
Stem cell research

Background

Induced cardiomyocytes are crucial for understanding heart development.
Lineage conversion is essential for cardiac repair strategies.
Subtype specification impacts sarcomere assembly.
Quantification of reprogrammed cells aids in mechanistic studies.

Purpose of Study

To generate diverse induced cardiomyocyte subtypes.
To quantify effects on cardiac subtype specification.
To explore regulatory cues in cardiac reprogramming.

Methods Used

Isolation of mouse embryonic fibroblasts from E 12.5 embryos.
Storage of cells in liquid nitrogen.
Harvesting and plating of plat-E cells.
Retrovirus-mediated delivery of Gata4, Hand2, Mef2c, and Tbx5.

Main Results

Successful generation of diverse cardiac subtypes.
Quantification of subtype-specific lineage conversion.
Insights into necessary cues for sarcomere assembly.
Foundation for further mechanistic studies.

Conclusions

This protocol enables the study of cardiac subtype specification.
It provides a framework for understanding cardiac reprogramming.
Future studies can build on these findings to enhance cardiac therapies.

Frequently Asked Questions

What is the main goal of this protocol?

The main goal is to generate and quantify diverse induced cardiomyocyte subtypes.

Why is subtype specification important?

Subtype specification is crucial for understanding heart development and repair mechanisms.

What cells are used in this study?

Mouse embryonic fibroblasts are isolated for reprogramming.

How are the reprogramming factors delivered?

Reprogramming factors are delivered using a retrovirus-mediated method.

What are the advantages of this technique?

It allows for quantified effects on cardiac subtype specification, aiding mechanistic studies.

What insights can be gained from this study?

Insights into the necessary cues for sarcomere assembly and subtype specification can be gained.

This manuscript describes a step-by-step protocol for the generation and quantification of diverse reprogrammed cardiac subtypes using a retrovirus-mediated delivery of Gata4, Hand2, Mef2c, and Tbx5.

The overall goal of this procedure is to generate and quantify a diverse set of induced cardiomyocyte subtypes. This method can help answer key, fundamental questions regarding subtype-specific lineage conversion during cardiac reprogramming such as the necessary cues that regulate sarcomere assembly and subtype specification. The main advantage of this technique is that it provides the method for quantified effects on cardio subtype specification which is required to perform mechanisis studies.

To begin, isolate mouse embryonic fiberblasts from stage E 12.5 embryos. Store them in liquid nitrogen as described in the accompanying text protocol. Next, harvest plat-E cells and plate them at one million cells per well into a six well plate so that the cells reach 70 to 80%confluency in 24 hours.

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