Researchers are looking for a new solution to control cells maturation


These days, scientists can collect a few skin or blood cells, wipe out their identities, and reprogram them to become virtually any other kind of cell in the human body, from neurons to heart cells.

The journey from skin cell to another type of functional cell involves converting them into induced pluripotent stem cells (iPSCs), which are similar to the developmentally immature stem cells found in embryos, and then coaxing them to mature into something different.

But the process runs on an invisible clock, one in which scientists are interested in speeding up so adult-like cells are available when needed, whether for testing drugs for precision medicine, transplanting to repair injury or defect, or better understanding basic biology.

It involves an FDA-approved compound called polyinosine-polycytidylic acid, or pIC, a double-stranded RNA molecule that activates a cell’s innate defense system.

The compound is commonly used to boost vaccines and chemotherapy.

The researchers found that when added to induced pluripotent stem cells undergoing the process of transitioning into cardiac muscle cells, pIC accelerated cellular maturation.

“We make beating heart muscle cells out of human iPSCs because we are interested in understanding and treating cardiac diseases,” says lead author and University of Wisconsin–Madison MD-Ph.D. student, Mitch Biermann.

“It’s important that the cells we make in a dish are as close to adult heart muscle function as we can make them.”

This is because, study leader Tim Kamp says, immature cardiac cells don’t contract as strongly as adult cardiac cells, and the electrical properties that sets their beat is different.

Their metabolic characteristics are a bit different, too.

“If you want to know how drugs, such as beta blockers, work in the adult heart, it’s better to test those in more mature, human iPSC-derived cardiomyocytes (cardiac muscle cells),” says Kamp, director of the University of Wisconsin–Madison Stem Cell and Regenerative Medicine and a professor of medicine in the School of Medicine and Public Health.

Other researchers have made progress utilizing a variety of ways to speed up the process, including electrical stimulation, changes to the metabolic environment of the cells, and even forcing the cells into the rod-like shapes more characteristic of adult cells, but each method seems to fall a bit short.

The discovery of induced pluripotent stem cells (iPSCs) by Takahashi et al[1] launched a novel field of medicine.

The ability to differentiate human iPSCs (hiPSCs) into various cell types allows for the generation of patient-, disease- and tissue-specific cells.

These cells enable precise disease modelling, in vitro drug testing, and clinical regenerative medicine approaches[1,2].

After a decade of research, iPSCs can now be successfully differentiated into hepatocytes[3], cardiomyocytes[4,5], neural cells[6,7], adipocytes[8] and many other cell types[9].

Cardiovascular disease is the greatest cause of mortality worldwide[10].

As such, modelling these diseases in vitro is of paramount importance to advance our understanding of disease and allow the development of new drug therapies.

Cardiomyocytes derived from human iPSCs (hiPSC-CMs) enable the creation of a patient-, heart-, and disease-specific in vitro model[5,11].

This is potentially most useful for the study of very rare cardiac disorders, including metabolic cardiomyopathies[12].

These hiPSC-CMs are remarkably powerful as they replicate the genome of the patient donor and allow characterization of various diseases and drugs in a non-invasive manner[2].

In addition, their ability to contract allows for characterization of contractility and can thus serve as an accurate and translatable cardiac drug model[13].

Recent studies have also showcased hiPSC-CMs’ ability to successfully engraft in a host organism[14,15].

One published study used macaque monkeys as a model for cardiomyocyte transplant outcomes. Transplanting human embryonic pluripotent stem cell derived-cardiomyocytes (hEPSC-CMs) through an intra-myocardial injection allowed the cells to graft with the host.

Once attached, these cells showed crucial electromechanical coupling with the host as demonstrated by echocardiography[15].

Similar regenerative medicine studies have also been performed using small (guinea pigs) and large (pigs) animal models[16,17].

Over the past few years, the efficiency of hiPSC-CM generation has been significantly improved.

Methods involving modulation of the GSK and Wnt pathways using small-molecule inhibitors have been widely used[5,18].

In addition, use of BMP and Activin A, along with the Matrigel sandwich method have proven successful[18].

Commercial kits such as those from STEMCELL Technologies (Vancouver, BC, Canada) and ThermoFisher Scientific (Carlsbad, CA, United States) have also entered the market and provide researchers with increased reproducibility and the ease of simplified protocols (Figure ​(Figure1).1).

Traditionally, hiPSC-CM generation has been characterised through flow cytometry staining for Troponin T (TNNT2), a cardiac-specific protein, in addition to visual qualification of spontaneously beating cell clusters.

Current protocols allow for the production of > 80%-90% TNNT2-positive hiPSC-CMs. This showcases the field’s success in achieving high-purity cardiomyocyte differentiation[2].

Through the use of lactate metabolic selection, > 99% TNNT2-positive cells have been successfully derived[19].

The derivation of highly-purified cardiomyocyte populations represented an important step forward for the field of cardiac regenerative medicine..

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Human induced pluripotent stem cell-derived cardiomyocytes. Immunocytochemistry for TNNT2 cardiac marker (green) and NKX2.5 early-mesodermal marker (orange). HiPSCs were differentiated into hiPSC-CMs using ThermoFisher’s Cardiomyocyte Differentiation kit. IPSCs were grown in a 6-well plate, differentiated into hiPSC-CMs for 14 d, fixed, stained, and then imaged using an epi-fluorescence microscope. Scale bar indicates 20 μm.

Although hiPSC-CMs are now being produced with high efficiency, an important problem remains.

The hiPSC-CMs generated with current protocols and commercial kits are qualitatively and quantitatively immature[2,20,21].

For example, in addition to immature calcium handling, hiPSC-CMs display immature ultrastructural and electrophysiological features, low expression of key maturation markers, and rely on glycolysis for their metabolism as opposed to fatty acid metabolism[2,20,22].

Immature cardiomyocytes have important differences when compared to adult cardiomyocytes and these differences may cause inaccurate disease modeling or drug testing and lead to unsuccessful clinical translation.

For example, the effect of cardiac drugs on contractile characteristics may be inaccurate when using an immature model.

However, given that hiPSC-CMs are being derived from pluripotent cells, it is not unexpected that the initial differentiated cells generated will be immature or fetal in their characteristics.

It is therefore reasonable to expect that an additional maturation protocol (Figure ​(Figure2)2) will be necessary to generate cells that truly reflect the in vivo tissue.

As such, many research groups are currently focussing on methods to promote the maturation of hiPSC-CMs so that they are suitable for accurate disease-modeling and clinical applications.

Methods evaluated to date include electrical stimulation, mechanical stimulation, modulation of carbon source, growth on various substrates, and the development of 3D culture conditions or organoids.

Studies have also shown the positive effect of prolonged culture time on hiPSC-CMs[23,24]; however, culturing hiPSC-CMs for >90 d is neither time- nor cost-efficient and, given that these cells are usually cultured without antibiotics, remains a fraught enterprise.

Therefore, other approaches must be used to create adult-like hiPSC-CMs within a reasonable time frame.

Current protocols for hiPSC-CM production have failed to mature these cells due to a lack of knowledge regarding the mechanisms of heart maturation in vivo.

At present, the field of cardiac regenerative medicine does not know the correct secretory factors, environmental cues, and external stimulation necessary to achieve proper adult-like cardiomyocytes.

Maturing hiPSC-CMs is key to fully realizing the potential of these cells. Without proper maturation, hiPSC-CMs could cease to be clinically relevant.

This review will examine the current methods for the maturation of iPSC-CM and suggest a way forward for the field.

Methods for the maturation of induced pluripotent stem cell-derived cardiomyocytes. A: Biochemical approaches involving the use of small-molecules and hormones, along with co-incubation with human mesenchymal stem cells; B: Environmental manipulation illustrated through the use of electrical stimulation to mature iPSC-CMs; C: 3-dimensional approaches showcased by Biowire, and 3D cardiac organoid generation in ECM layered tissue-culture plates.

Biermann chose a different tack.

He noticed that cardiac cells derived from iPSCs mature at different rates in a dish.

Other researchers found that cardiomyocytes in the heart and in blood vessels of rats matured according to the same clock, despite being distant from each other in the body.

“Maturation isn’t only controlled by what’s going on in the environment of the heart,” Biermann says.

“Because of that and because maturation in a dish seems random, we started thinking about epigenetics.”

In other words, Biermann thought maturation rates might have to do with the way timing of cellular maturation events was coordinated.

He wondered whether he could essentially prime the cells at just the right time to accelerate maturation – to wind up the clock – and began looking for compounds that did so without also killing them.

That’s how he found pIC.

When he added it to early cardiac precursor cells in the lab, they formed beating heart cells two days sooner than cells without pIC.

After 48 hours, the cells were removed from the compound but its effects continued to linger, leading to cells that were larger in size, had better contractility, were electrically more efficient, exhibited mature metabolic characteristics and had better-developed structures when compared to cells without pIC.

When they looked closely at what was going on inside the cells exposed to the compound, they found that pIC had activated cellular programming that led to accelerated maturation.

Specifically, it turned up the expression of the JAG1 gene (which triggers a signaling pathway called Notch), and led to a host of epigenetic changes.

The researchers also found that early cardiomyocytes exposed to pIC before implantation in mouse hearts matured faster than those not primed with the compound.

They think pIC makes the cells more receptive to the maturation cues already present.

“There is some intrinsic clock function involved as well, which, in part, is based on epigenetic changes,” says Kamp. “It’s safe to say there is much more to learn that we don’t yet understand about cell autonomous developmental clocks.”

Developmental clocks, researchers think, dictate the amount of time it takes for a fertilized egg to develop from a single immature cell into a newborn possessing all the specialized cells of the body.

Despite being composed of the same cellular stuff, a baby mouse takes 21 days to develop, a human about 280 days, and an African elephant 600 days or more.

Biermann’s finding, Kamp says, was a surprise, because no one has thought about using pIC or compounds like it for this application.

It also presents an opportunity to combine with other methods for accelerating maturation, and for doing so at a larger scale since it can be easily added to and washed out of cells. But the finding is also not without caveats.

“One obvious question is whether this is cardiomyocyte specific or if it could be useful in making neurons of pancreatic islet cells (defects in which can lead to diabetes),” says Kamp.

He also points out that these accelerated cardiomyocytes are still not an exact match for adult heart muscle cells.

“We are not at the promised land yet,” he says. “We haven’t seen any aberrant effects, but we don’t know.”

Further, they don’t yet know how these cells will continue to age.

“If we’re turning up the clock, are they going to age faster, too?” explain Kamp. “If we can get a better handle on it, it could be used for practical purposes and for better understanding development.”

Provided by University of Wisconsin-Madison


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