Here, we present a protocol to culture pharyngeal arches to study the biology of heart and muscle progenitor cells and their microenvironment.
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.
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 developmental processes including induction, proliferation, and differentiation of head and heart progenitors in the pharyngeal mesoderm are under active investigation 5-7.
Until recently, it remained unknown whether cardiac progenitor cells undergo expansion without differentiation, partly due to the lack of information on their cellular environment. Our recent study suggests that pharyngeal arches serve as a microenvironment for the renewal of cardiac and muscle progenitors and can be cultured ex vivo over several weeks 8. This explant method offers a novel and unique opportunity to study the development of cardio-craniofacial progenitors ex vivo.
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
2. Surgical Procedures
3. Incubation and Imaging
During development, facial muscle and heart progenitors can be traced as they proliferate and migrate from the 1st and 2nd 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 of culture. Within 3 days of culture myotube formation can be observed from 1st pharyngeal arches and cardiomyocyte formation from the 2nd pharyngeal arches (Figure 2A and 2B). Cardiomyocyte formation can be visually confirmed by spontaneously contracting clusters of migrating cells and/or analysis of specific cardiomyocyte markers (e.g., Cardiac Troponin T). Myotube/facial muscle formation can be visually observed by long elongated (up to 500 µm in length) spontaneously twitching cells and/or analysis of specific muscle markers (e.g., Myogenin). By using the cre-lox system in mice, mesoderm progeny can be traced by fluorescent reporters upon cre-expression during ex vivo culture (Figure 2A’ and 2B’). Likewise, with this method it is possible to study the fate of progeny wherein a specific gene of interest is conditionally knocked out or overexpressed, which otherwise would result in early embryonic lethality as we previously described 8.
Figure 1: Mouse embryo (Mesp1cre, Ai9) 9.5 days post fertilization (E.9.5). (A) Left side of embryo. (PA: Pharyngeal Arch, OT: Outflow tract, LV: Left ventricle. (A’) Mesp1 linage-trace; RFP marks Mesp1+ cells and their progeny. Arrow indicates RFP+ Mesp1-progeny in PA2 that is continuous with the OT. (A’’) Dashed line indicate where to cut PA1 and PA2 posterior to the heart. (B) Butterfly-like right and left PA1 and PA2 after dissection from the embryo. FE: Foregut endoderm. (B') RFP marks Mesp1 progeny in PA1 and PA2. (B’’) * and ** along with the dashed lines indicates where to cut and release PA1 and PA2 from the OT and FE. Scale bar: 500 µm. Please click here to view a larger version of this figure.
Figure 2: Cultured pharyngeal arches. (A) Cultured PA1 (3 days of culture). (A’) RFP marks Mesp1 progeny in PA1. Arrows and * indicate early myotube formation. (A’’) Overlay of RFP expression in PA1. (B) Cultured PA2 (8 days of culture). (B’) RFP marks Mesp1 progeny in PA2. Arrows and ** indicate beating area of cardiomyocytes (modified from Shenje et al. 8). (B’’) Overlay of RFP expression in PA2. Scale bar: 250 µm. Please click here to view a larger version of this figure.
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 advanced mouse embryo dissection skills. Once arches have been successfully dissected using this stepwise protocol and transferred to pre-coated wells, it is critical that arches attach to the bottom area. In this protocol, we coat wells with 10% FBS followed by culture in SFM resulting in attachment and growth of >75% of arches, however other coating materials can be used (matrigel, gelatin, fibronectin etc.) as well as serum containing media.
The pharyngeal arches serves as primordia for a multitude of structures during development. After initial formation of the pharyngeal arches (8 days post fertilization in mice), CPCs expand in the PA2 and migrate into the cardiac outflow tract where they differentiate into cardiac cells, thereby serving as a renewable source of CPCs that supplies cells needed to sustain heart growth. As the pharyngeal arches are temporal developmental structures (E8-11.5) and migration of CPCs into the outflow tract occurs from approximately E8-E10) this brings certain limitations to the technique. For CPC studies, we recommend using pharyngeal arches dissected between E9-10, at this stage each PA2 contain approximately 5-800 RFP+ cells (Mesp1 linage trace).
With fluorescent labeling, this ex vivo culture allows us to monitor developing progenitors and their differentiation into beating cardiomyocytes or myotubes in real-time. In addition, this ex vivo culture system can be used to study roles of cell-autonomous and microenvironmental factors affecting cardiac/muscle progenitor cell proliferation, migration, and differentiation using existing Cre/loxP lines. As such, this system is expected to facilitate cardiac/muscle progenitor–niche studies, which may lead to a better understanding of heart/muscle formation and disease.
The authors have nothing to disclose.
We thank Kwon laboratory members for helpful discussions. This work was supported by grants from NHLBI/NIH (R01HL111198) and Maryland Stem Cell Research Fund (MSCRF).
FBS | Hyclone | SH30071.03 | |
PBS + Ca2 + Mg2 | Corning | 21-030-CV | |
10cm 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) à (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 |