A 3D human model for studying Kleefstra syndrome

How can we study in the laboratory what happens to the developing brain when the EHMT1 Is it not working normally?;

A research team from Hungary has created a new three-dimensional human cell model of Kleefstra syndrome, using cells derived from an individual with the syndrome.

The study entitled Establishment of human pluripotent stem cell-derived cortical neurosphere model to study pathomechanisms and chemical toxicity in Kleefstra syndrome published in Scientific Reports and describes the creation and characterization 3D cortical neurospheres, meaning small three-dimensional structures of nerve cells grown in the laboratory that can be used to study the development and function of the human nervous system.

The most important result of the work is not that a cure was found.

It is that a was created human laboratory model of Kleefstra, which replicates several of the cellular and molecular characteristics that have been linked to EHMT1 deficiency and may in the future be used for investigating disease mechanisms and for screening potential drug compounds.

What are iPSCs

The study was based on a technology that has significantly changed the way rare genetic disorders are studied.

The researchers used induced pluripotent stem cells, iPSCs, into induced pluripotent stem cells.

Simply put, cells are taken from a human and, through a special laboratory process, «reprogrammed» to regain the ability to develop into different types of cells.

They can then be directed to convert, for example, into nerve cells.

The big advantage is that these cells they retain the genetic background of the human from whom they originated.

In the case of this study, the Kleefstra cell line was derived from an 11-year-old girl with a pathogenic variant of EHMT1, which led to the loss of gene function. The researchers also used cells from a healthy individual as a control group.

From human cells to 3D neurospheres

Initially, the iPSCs were converted into neural progenitor cells, meaning cells that are at an early stage and can develop into different types of nervous system cells.

Then the researchers cultured them in a three-dimensional format to create cortical neurospheres, small spherical structures composed of nerve cells and having characteristics related to the cerebral cortex.

Neurospheres were cultured for 57 days, allowing researchers to track their development and compare the Kleefstra model with the control model.

This is not a complete «minibrain».

Neurospheres are much simpler than the human brain and even the most complex brain organoids. However, this relative simplicity is also an advantage, because neurospheres can be more homogenous and reproducible, something particularly useful when we want in the future to compare many different substances in a drug screening system.

Differential development of Kleefstra neurospheres

One of the first visible differences concerned the way three-dimensional structures were developed.

Neurospheres were formed from both the cells of the individual with Kleefstra and the control cells.

However, while the neurospheres of the control group continued to increase in size during culture, The development of Kleefstra neurospheres was significantly restricted.

At the end of the 57 days, their size remained approximately at the level from which the three-dimensional culture had started.

Researchers simultaneously identified lower expression of Ki67, a cell proliferation marker, as well as changes in other markers related to cell growth and adhesion.

They also confirmed lower levels H3K9me2, an epigenetic modification normally associated with EHMT1 activity.

This finding is important because it shows that the laboratory model maintains a basic biological consequence of the deficiency of EHMT1.

More neurite outgrowth

Despite the limited growth of the neurospheres themselves, a result appeared that at first might seem contradictory.

When the researchers examined the neurite outgrowth, meaning the extensions created by nerve cells through which neuronal connections gradually form, Kleefstra cells showed greater growth than those of the control group.

At the same time, an increased expression of was detected BDNF, a major neurotrophic factor involved in the development, survival, and plasticity of neurons.

These results show that the effect of EHMT1 deficiency on neurodevelopment is not simply a generalized «reduction» in development.

Different processes seem to be changing in different ways.

And this is precisely one of the advantages of such human cellular models. They allow researchers to examine these processes separately and try to understand how they are connected to each other.

Different composition of neuronal populations

Another significant difference concerned what types of neurons developed inside the neurospheres.

In the Kleefstra model, there was a greater presence of neurons expressing VGLUT1/2, markers related to glutamatergic neurons.

Also increased were cholinergic neurons, who were identified through ChAT expression.

Instead, the TH-positive catecholaminergic neurons they were significantly fewer and almost absent from the Kleefstra neurospheres compared to the control model.

These results indicate that EHMT1 deficiency may affect not only the development of nerve cells but also the way in which they differentiate towards specific neuronal populations.

NMDAR1 appears again

Another finding of particular interest.

The researchers found increased expression of NMDAR1 in Kleefstra neurospheres.

NMDAR1 is a key subunit of NMDA receptors, which are involved in glutamatergic neurotransmission and play an important role in synaptic plasticity, learning, and the development of neuronal networks.

The finding becomes more interesting because alterations in NMDA receptor function had also been observed in previous laboratory studies using neurons derived from individuals with Kleefstra.

The authors therefore consider that the increased NMDAR1 expression may represent a recurrent feature of Kleefstra human neuronal models.

This does not mean that it has already been proven that NMDAR1 is a therapeutic target in humans.

However, it shows a biological mechanism that appears in more than one laboratory model and therefore deserves further investigation.

Why did the researchers also examine chemical sensitivity

A second part of the study concerned the possibility that EHMT1-deficient cells react differently to external chemical influences.

The researchers exposed both neural progenitor cells and mature neurospheres to four different substances:

paraquat, rotenone, bardoxolone and doxorubicin.

The goal was not to examine these substances as treatments for Kleefstra.

They were used as laboratory tools to investigate whether the genotype and developmental stage of neural cells affect their susceptibility to different chemical agents.

The results showed that the sensitivity indeed depended both on the substance and on the developmental stage of the cells.

Especially the mature Kleefstra neurospheres exhibited greater sensitivity to paraquat.

The finding is of research interest, but it should not be translated into a clinical conclusion that individuals with Kleefstra are generally more sensitive to specific environmental substances.

The study was conducted in vitro, in cultured cells, and much more research is needed before such observations gain any significance for the daily care of individuals with the syndrome.

Why such a 3D model is important

In order for a targeted treatment for a genetic neurodevelopmental disorder to eventually be developed, it is not enough to know which gene is responsible.

We need to understand what exactly changes inside human cells when this gene does not function properly.

In Kleefstra, we know that the loss of function of EHMT1 alters the epigenetic regulation of many other genes.

The next question is how this initial genetic alteration leads to the changes observed in development, neuronal function, and ultimately the phenotype of the syndrome.

Animal models remain extremely important, but they cannot fully replicate the development and function of the human brain.

On the other hand, traditional flat-surface cell cultures cannot reproduce the three-dimensional cell-cell interactions that exist within a tissue.

Ta patient-derived 3D models They are trying to bridge this gap.

From the study of mechanisms in drug screening

Perhaps the model's greatest future value is that it can be used as screening platform.

Once a laboratory model displays repeatable characteristics of Kleefstra, researchers can in the future examine whether a substance can alter these characteristics.

For example, it could be examined whether an experimental intervention:

reduces or restores an abnormal molecular pathway,

improves the development of nerve cells,

restores more normal neuronal differentiation,

or alters other measurable markers associated with EHMT1 deficiency.

Because neurospheres are relatively homogeneous and can be produced in larger numbers, they constitute a system that may be suitable for testing of many different candidate compounds. The authors themselves cite drug development and screening toward more personalized therapeutic strategies as potential applications of the model.

This is not yet a cure

This particular distinction is very important.

The study did not test a treatment for Kleefstra syndrome and did not show that any pharmaceutical substance can improve the characteristics of the syndrome in humans.

The substantial achievement is the creation of the model itself.

Before we can systematically evaluate hundreds or thousands of potential compounds, we need a reliable laboratory system in which to test them.

This specific work is a step towards the creation of such an infrastructure.

A major limitation, the model comes from a single person

Despite the interesting results, there is a very important limitation.

The Kleefstra model was created by a patient-derived iPSC cell line, i.e., from an individual with a specific pathogenic variant of EHMT1.

Kleefstra syndrome exhibits significant genetic and clinical heterogeneity.

Different people may have different sequence variants in EHMT1 or larger deletions of the 9q34 region, while even individuals with similar genetic alterations may present with a different phenotype.

Therefore, we do not yet know to what extent all the features observed in this cell line are common across the entire Kleefstra spectrum.

The creation of corresponding 3D models from more people with different genotypes it will be particularly important for the confirmation of the findings.

From Kleefstra's description to its biology

For many years, research into Kleefstra syndrome focused mainly on gene identification, the description of clinical characteristics, and a better understanding of the phenotype.

These studies remain essential.

At the same time, however, a different type of research is now developing.

The ability to create human nerve cells carrying the Kleefstra genetic mutation and organize them into three-dimensional systems gives researchers a new tool to answer these questions.