PANDAS Neurologic Stem Cell Research, iPSC Modeling
Understanding the Role of Genetic Risk Factors for PANDAS Using
Human Induced-Pluripotent Stem Cells and Disease Modeling
A PANDAS Network $50,000 grant to the Irene Munk Pedersen Laboratory,
Scintillon Research Institute (San Diego, CA) received a grant to conduct a pilot study examining the direct effects of illness on the brains of children with PANDAS/PANS. The Pedersen Laboratory conducts stem cell research using induced pluripotent stem cells (iPSCs) to generate various brain cell types and develop novel 3D brain-chip culture models. This has led to research that deepens our understanding of how the blood-brain barrier, brain immune cells, and neuronal cells may function together during active disease. iPSC research has been around for twenty years and is a pivotal approach for modeling a variety of neurologic diseases such as multiple sclerosis, ischemic stroke, Alzheimer’s disease, and more. Advancements in iPSC modeling have enabled scientists to observe the progression of neurological conditions at the cellular level, uncovering new biomarkers and therapeutic targets.
At Columbia University, in Agalliu’s lab, blood samples from three PANDAS
patients and three controls (non-PANDAS) were selected to use in this unique in vitro 3D brain chip culture model examining aspects of the brain environment during disease. Sometimes this is called studying a “disease in a dish” because the stem cells (iPSCs) are derived from patients’ cells and then differentiated into brain cells that function similarly to cells in the human brain. In the first experiment, we studied the effect of PANDAS blood serum on the blood-brain barrier (BBB).
The image above shows a significant effect on BBB permeability when brain endothelial cells interact with inflammatory cytokines in the blood, compared with the control. One of the measurements taken was examining if a green protein in a fluid called a tracer was used to leak through the BBB (top panels), and imaging using a
microscope revealed that tight junction proteins had formed gaps and zigzags between the cells of the blood-brain barrier, making it leaky and allowing immune cells to cross the BBB and enter the brain.
The Agalliu lab narrowed down “10 risk genes” that are expressed in neurons,
microglia and macrophages, all of which are important in both synaptic transmission and immune cell function.
Using the 3D brain-chip model, it is possible to look at both BBB permeability,
BBB supporting cells (astrocytes and pericytes), glutaminergic neurons and brain
specific immune cells (microglia); in other words, it is possible to study the full 3D
BBB/CNS to better understand how interactions between brain cells, such as neurons and immune cells, affect brain function. Looking at the shape of cells, a term called “morphology”, can serve as a window into the function of cells, which can be affected by both genetics and environmental factors.
Next, we wished to better understand how a leaky blood-brain barrier may affect
neurons within the brain, if a damaging component in the blood is allowed to enter the CNS. To address this question, we examined mature neurons that we had exposed to sera from control or PANDAS children. Neurons were studied either alone or with other, supportive brain cells called astrocytes and microglia. (Definitions of these cells are: astrocytes function to maintain brain homeostasis, including blood flow and neuronal synaptic transmission. Microglia function as primary immune cells to the CNS and scan for “debris” and repair of neuronal circuits.)
In this observation, it was unclear whether glutaminergic neurons were more affected in cultures with PANDAS sera than in cultures with control sera. It is possible that the normal sera would not reach the neurons because control cases do not have a leaky BBB. However, the experiment shows that a component in the patient’s blood can alter the shape of neurons compared with neurons not exposed to sera.
In the images of the tri-culture exposed to sera, we observed a cluster of neurons
forming a shared “nucleus” of neuronal cell bodies, often observed when neurons are in a stress situation. The cultures NOT exposed to PANDAS sera showed neurons with separate cell bodies that were close together, likely allowing enhanced neuronal signaling that supports the brain. In addition, cultures exposed to either PANDAS sera or control sera demonstrated a reduction in the number of astrocytes, which normally support neurons. Finally, serum exposure increased the number of microglia, which respond to stressful environments in the brain. This needs to be studied with a larger cohort to determine if, in fact, there is a destructive cellular environment that could be part of the brain.
In summary, we find that sera from some PANDAS children contain components that induce BBB leakiness, allowing CNS cell exposure and potentially leading to neuronal toxicity, astrocytic loss, and microglial activation.
