Ex Vivo Culture of Pharyngeal Arches to Study Heart and Muscle Progenitors and Their Niche

Summary

Here, we present a protocol to culture pharyngeal arches to study the biology of heart and muscle progenitor cells and their microenvironment.

Abstract

The pharyngeal mesoderm of developing embryos contributes to broad regions of head and heart musculature. We have developed a novel method to study head and heart progenitor cell development with pharyngeal arches (also known as branchial arches) ex vivo. Using this method, we have recently described that the second pharyngeal arch contains self-renewing heart progenitors and serves as a microenvironment for expansion of the progenitors during mouse heart development. The progenitor cells remain undifferentiated and expansive inside the arch, but quickly become functional cardiomyocytes as they migrate out of the arch. We also reported that first pharyngeal arch contains muscle progenitors giving rise to myotubes after leaving the arch. Here, we demonstrate the procedure for the dissection and ex vivo culture of first and second pharyngeal arches from developing mouse embryos. The method enables one to study head and heart progenitor/muscle development, including cardiomyocyte and myotube formation in detail ex vivo.

Introduction

Pharyngeal mesoderm cells give rise to parts of the heart and the pharyngeal muscles. During embryonic development, multipotent cardiac progenitor cells from the second heart field migrate from the pharyngeal mesoderm and populate the cardiac outflow tract and right ventricle, and their abnormal development is closely associated with congenital heart disease—the leading cause of birth defects and birth defect-related deaths in humans ^1-3. Recent studies have demonstrated that pharyngeal mesoderm contributes to head muscles, in addition to the heart, making the mesoderm a critical part of the cardio-craniofacial development ⁴. Thus, the deve....

Protocol

All mice were maintained at an American Association for the Accreditation of Laboratory Animal Care (AAALAC)–accredited animal facility at the Johns Hopkins University and housed in accordance with the procedures outlined in the Guide for the Care and Use of Laboratory Animals. The Institutional Animal Care and Use Committee (IACUC) approved all experimental protocols.

1. Experimental Preparation

1. Coat 12-well plate with 10% Fetal Bovine Serum (FBS) in Phosphate Buffered Solution (PBS) for 60 min.
2. Remove coating solution and add 200 µl of Serum-free Media (SFM). Twirl plate to make sure that a film of media covers the entire surf....

Results

During development, facial muscle and heart progenitors can be traced as they proliferate and migrate from the 1^st and 2^nd pharyngeal arch, to become head and heart musculature, respectively (Figure 1A, A’ and A’’). Culturing pharyngeal arches offers a unique way to study heart and muscle development in detail ex vivo. After dissection and attachment of pharyngeal arches, migrating cells from attached arches can be observed within 24-48 hr .......

Discussion

In this video, we demonstrate how to isolate and culture first and second pharyngeal arches of 9.5 days old mouse embryos. Pharyngeal arches are transient, segmented bulges that appear on the craniolateral side of developing embryos ⁹, which contain multi-potent cardiac progenitor cells—building blocks to make the heart during embryogenesis ^10,11—in second arches and head muscle progenitors in first arches ⁸.

Dissection of pharyngeal arches requires.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
FBS	Hyclone	SH30071.03
PBS + Ca2 + Mg2	Corning	21-030-CV
10 cm Petri plates	Fishersci	FB0875712
Scissor
Dissection forceps
Serum Free Media:
IMDM	Cellgro	15-016-CV
Ham’s F12	Cellgro	10-080-CV
N2-SUPPLEMENT	Gibco	17502-048
B27-retinoic acid	Gibco	12587-010
10% BSA (in PBS) (Invitrogen Cat#. P2489)	Life Sciences	P2489
100x Glutamine (Gibco Cat.# 25030-081)	Gibco	35050-61
100x Pen/Strep (Gibco Cat#. 15070-063)	Gibco	15140-122