Long QT syndrome, also referred to as LQTS, is a genetic disorder which affects the repolarization of the heart after a heartbeat. This prolonged repolarization of the heart is observed as a lengthening of the QT interval in the electrocardiogram, hence the disorders name. A long QT interval can upset the careful timing of the heartbeat and trigger dangerous heart rhythms which can result in fainting, seizures, or sudden death. In this webinar, Dr. Vincenzo Macri (STEMCELL Technologies) discusses how the stem cell culture and gene editing technologies being developed by his lab can be used to recreate these patient heart beats in a dish.
Transcript of the webinar
Thank you for joining us for today's coffee break webinar.
Today's topic is recreating irregular heartbeats with patient cells and gene editing The human heart typically beats 80 times in a minute 115200 times per day or in other words 42 million times a year. So if you live to be 80 years old your heart would have beaten approximately three billion times. The arhythmic beating of the heart is attributed to the interplay of numerous ion channels expressed in the cardiac muscle, these ion channels underlie the excitability also known as the action potential which initiates cardiac muscle contraction. Long QT syndrome also referred to as LQTS is a condition which affects the repolarization of the heart after a heartbeat. This prolonged repolarization of the heart is observed as a lengthening of the QT interval in the electrocardiogram hence, the disorders name LQTS can arise from the mutation of one of several genes a common type of LQTS, LQTS 2 arises from a mutation in the human Ether-a-go-go Related Gene potassium channel (hERG), which is responsible for curtailing the cardiac action potential a long QT interval can upset the careful timing of the heartbeat and trigger dangerous heart rhythms which can result in fainting seizures or sudden death these episodes are often triggered by exercise or stress.
In this webinar Dr.Vincenzo Macri senior scientist at stem-cell technologies demonstrates how the stem cell culture and gene editing technologies being developed by his lab can be used to recreate these patient heartbeats in a dish
Thank you for the introduction Melissa as mentioned earlier I'll be discussing how we can create irregular heartbeats with patient cells and gene editing making human pluripotent stem cell-derived cardiomyocytes is technically challenging stem cell has developed a media kit so you can efficiently make human pluripotent stem cell-derived cardiomyocytes in your lab. Our stem diff cardiomyocyte differentiation kit can take high quality human pluripotent stem cells and differentiate them to a confluent monolayer of beating cardiomyocytes within 15 days our differentiation kit produces greater than 80% CT+T positive cells and one kit can generate well over 50 million cardiomyocytes our stem diff cardiomyocyte system consists of a differentiation kit, maintenance kit, and dissociation kit.In this webinar, I will present data showing how our stem diff cardiomyocyte system and Maestro MEA system can be used to model long QT Type 2 syndrome.
Microelectrode array can be used to measure the excitability of cardiomyocytes, the excitability is measured as a field potential the field potential is similar to an action potential observed at the single-cell level and similar to a clinical ECG. They share some similar phases for example sharp upstroke or depolarization phase, after the depolarization phase beat timing and arrhythmias can be measured quite easily in a microelectrode array. We use the microelectrode array system to track the performance of HPSC differentiation to cardiomyocytes using a 48 well CytoView plate we seeded down human pluripotent stem cells into each well of the plate, we then differentiated those cells and tracked excitability in real time from days 8 to 26 of the differentiation and maintenance protocol.
Using our stem diff cardiomyocyte differentiation kit we were able to successfully track excitability using the Maestro MEA system during the HPSC differentiation process to cardiomyocytes as well as maintenance of those cardiomyocytes, using two ES and two IPS lines we observed the onset of consistent excitability metrics at day fourteen. Eighty-three two hundred percent of the wells contain regular beating cardiomyocytes with very consistent excitability profiles up until day twenty-five. Variability across those lines were small with respect to the beat period and a few potential duration and we saw less than 10% beat period irregularity across the 4 hPSC lines.
In In the previous slide, we showed that using the stem diff cardiomyocyte differentiation kit produces hPSC derived cardiomyocytes with reliable and consistent excitability profiles across multiple hPSC lines we next want to model a long QT type to syndrome which is due to a mutation in a hERG channel, the hERG potassium channel is required for cardiac repolarization, figure A shows a ventricular action potential the ventricular action potential begins with a rapid upstroke or depolarization phase followed by a slow repolarization phase the hERG potassium current is an outward current shown in blue when there's a mutation in the hERG channel the outward her current can be reduced shown in red the reduction in the hERG current results in a prolongation of ventricular action potential in Figure C is a surface electrocardiogram when there is a mutation in the hERG channel which produces less outward hERG current we can see that this shows a prolongation of the QT interval it can result in the onset of Torsades de pointes.
To disrupt the hERG potassium current we used a CRISPR Cas9 gene editing system to target the past domain which is located in the end terminus of the hERG potassium channel we created an IPS line that carried a four nucleotide deletion in the past domain which resulted in an early stop codon, we next generated cardiomyocytes using our control and hERG edited isogenic hPSC lines using our stem diff cardiomyocyte differentiation kit. On day 18 we used our stem diff cardiomyocyte dissociation kit to harvest and re-plate the cardiomyocytes into the CytoView MEA 48 well plate. On day 28 we took our first measurements of the field potential signals from both the control and hERG edited hPSC derived cardiomyocytes, here we show images of the control and hERG edited hPSC derived cardiomyocytes on day 28 in a CytoView MEA plate. Next to the images our field potential signals recorded from the hERG edited and control hPSC derived cardiomyocytes we can observe that the hERG edited hPSC derived cardiomyocytes have a prolonged FPD and beat period compared to the control the hERG edited cardiomyocytes show an expected increase in FPD as a result of the heterozygous mutation in the hERG channel. Here we show field potential recordings for the control and the hERG edited hPSC derived cardiomyocytes for a 45 second period we can observe that the control hPSC derived cardiomyocytes as stable and consistent field potential signal however for the hERG edited hPSC derived cardiomyocytes we observe the delay in repolarization a variability in beat period but also the onset of spontaneous rapid beating which resembles Torsades de pointes.
We next want to model the long QT type 2 syndrome using patient-derived IPS lines and the stem diff cardiomyocyte system. We obtained the IPS lines from the Frasier lab at UCSD, this family has a a heterozygote point mutation in a distal C terminus of the hERG ion channel which results in an early stop codon. We obtained the unaffected grandmother to 6 and the affected granddaughter to 1 here we show images of the hPSC derived cardiomyocytes generated from the wild-type and the long QT type 2 patient arrived IPS lines on day 28 of our differentiation and replating protocol next to the images are the few potential signals recorded from the hPSC-derived cardiomyocytes we can observe that a 2 6 control line or wall type line has a stable and predictable excitability profile, however, to one who is a carrier of the heterozygote point mutation and exhibits long QT type 2 has a prolonged FPD compared to the wild type and a prolonged beep period.
During this webinar we've shown that the long QT type 2 syndrome can be modeled using IPS lines that have different genotypes, the future of these clinical trials in a dish lends itself to experiments where one could predict drug response and if based on the patient's genotype, therefore we can use a precision medicine approach to guide the drug selection for the patients based on their genetic background.
In summary, I've showed you that the CRISPR cast nine gene-editing system can be used to target hERG to produce hPSC- derived cardiomyocytes with the expected long QT phenotype. Cells derived from patients with long QT also produce hPSC- derived cardiomyocytes with the expected phenotype. For example the prolongation of the field potential duration, the stem diff cardiomyocyte system is efficient and robust method to produce many hPSC- derived cardiomyocytes that can be used for disease modeling and the Maestro multiwell MEA system allows for the tracking of hPSC cardiomyocyte differentiation and excitability over hours days and weeks label-free and in real time.
I would like to conclude this coffee webinar by acknowledging the groups of people who have contributed to this work at stem cell or at R&D Marketing Group and i particular Adam Hirst and Jessica Norburg and from Axion Biosystems Stacey Chavatl and Mike Clements, thank you for listening.
And that is the conclusion for today's coffee break webinar, if you have any questions you would like to ask regarding the research presented or if you are interested in presenting your own research with microelectrode array technology please forward them to coffee break@axionbio.com. For questions submitted for Dr. Macri, he will be in touch with you shortly. Thank you for joining in on today's coffee break webinar and we look forward to seeing you again!
