Generation of Murine Cardiac Pacemaker Cell Aggregates Based on ES-Cell-Programming in Combination with Myh6-Promoter-Selection

Summary

This protocol describes how to produce functional sinus nodal tissue from murine pluripotent stem cells (PSC). T-Box3 (TBX3) overexpression plus cardiac Myosin-heavy-chain (Myh6) promoter antibiotic selection leads to highly pure pacemaker cell aggregates. These “Induced-sinoatrial-bodies” (“iSABs”) contain over 80% pacemaker cells, show highly increased beating rates and are able to pace myocardium ex vivo.

Abstract

Treatment of the “sick sinus syndrome” is based on artificial pacemakers. These bear hazards such as battery failure and infections. Moreover, they lack hormone responsiveness and the overall procedure is cost-intensive. “Biological pacemakers” generated from PSCs may become an alternative, yet the typical content of pacemaker cells in Embryoid Bodies (EBs) is extremely low. The described protocol combines “forward programming” of murine PSCs via the sinus node inducer TBX3 with Myh6-promoter based antibiotic selection. This yields cardiomyocyte aggregates consistent of >80% physiologically functional pacemaker cells. These “induced-sinoatrial-bodies” (“iSABs”) are spontaneously contracting at yet unreached frequencies (400-500 bpm) corresponding to nodal cells isolated from mouse hearts and are able to pace murine myocardium ex vivo. Using the described protocol highly pure sinus nodal single cells can be generated which e.g. can be used for in vitro drug testing. Furthermore, the iSABs generated according to this protocol may become a crucial step towards heart tissue engineering.

Introduction

The term “sick sinus syndrome” summarizes multiple diseases leading to the deterioration of the cardiac pacemaker system. It comprises pathological, symptomatic sinus bradycardia, sinoatrial block, sinus arrest as well as the tachycardia-bradycardia syndrome. Thereby, a “sick sinus syndrome” is often accompanied by general cardiac diseases such as an ischemic heart disease, cardiomyopathies or myocarditis. At present, therapeutic approaches are based on the implantation of electrical pacemakers. However, this goes along with a number of risks such as infections and battery failure. Overall, the incidence of complications is still very high in p....

Protocol

1. Recommendations Before Starting

1. Do not use PSCs contaminated with mycoplasma because they will not differentiate properly into sinus node cells. Test for mycoplasma contamination before starting the protocol. Do this using a PCR kit for rapid, highly sensitive detection of mycoplasmas and follow the manufacturer`s protocol.
2. For each Petri dish (step 2.3.4), coat one 10 cm² cell culture dish with sterile 7 ml 0.1 % gelatin from cold water fish skin for 1 hr at 37 °C. Remove the gelatin and let the dish dry under sterile conditions in a sterile bench.
3. Before you can start the differentiation protocol you need a double t....

Results

The described protocol allows generation of iSABs with a beating frequency of around 450 bpm from PSCs (shown in the movie) which is near to the mouse heart beating frequency. After dissociation of iSABs (step 2.8.8) the observed single cells show the typical shape of cells of the sinus node (spindle and spider cells) as shown in Figure 1. These cells highly express proteins which are known to be essential for the function of the sinus node like hyperpolarization-activated cyclic nucleotide-gated cation .......

Discussion

The ability to produce stem cell derived cardiac pacemaker cells may allow reconstitution of the proper cardiac rhythm in the sense of “biological pacemakers”. Likewise, drug testing in vitro will benefit from their availability. PSCs can give rise to any cell type of the mammalian body including cardiomyocytes with pacemaker cell properties^{8,9,10,11,12,13}. However, typically the cell populations within “Embryoid Bodies” are highly heterogeneous, which inevitably leads to the requiremen.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Iscove's liquid medium with stable glutamine	Biochrom AG	FG 0465
DMEM liquid medium without Na-pyruvate, with stable glutamine	Biochrom AG	FG 0435
CLS type IV, CLS IV	Biochrom AG	C4-22
Non-essential amino acids	Biochrom AG	K 0293
FBS Superior	Biochrom AG	S 0615
Sodium pyruvate (100 mM)	Biochrom AG	L 0473
G 418-BC liquid (ready-to-use solution), sterile	Biochrom AG	A 2912
Reagent reservoir PP f.multichannel pip. 60ml,sterile	Brand	703409
Accutase	eBioscience,Inc.	00-4555-56
Eppendorf Xplorer/Xplorer plus, electronic pipette	Eppendorf	4861000155
Falcon Cell Strainer 40 µm	Falcon	352340
Penicillin/Streptomycin	GE Healthcare	P11-010
Petri Dishes	Greiner BioOne	663102
DPBS without Ca and Mg	PAN-Biotech	P04-36500
Fetal bovine serum	PAN-Biotech	P30-3302
Leukemia inhibitory factor	Phoenix Europe GmbH	LIF-250
Quadratic petri dishes	Roth	PX67.1
Gelatin from cold water fish skin	SigmaAldrich	G7765
2-Mercaptoethanol	SigmaAldrich	M3148
1-Thioglycerol	SigmaAldrich	M6145
Tissue culture dishes	TPP	93100
Tissue culture flask	TPP	90076
Tissue culture test plates (24 well)	TPP	92424