Scientists were able to develop a three-dimensional model of
six cell types — neurons, astrocytes, microglia, endothelial cells, pericytes,
and oligodendrocytes. The spheroids have all the qualities of blood-brain
barrier function, and will make it easier to test new drugs in the pipeline and
their potential for neurotoxicity.
Scientists at Wake Forest School of Medicine have created a
model for the human blood-brain barrier (BBB) in the laboratory, a technical
feat that could make it easier to understand how it works to protect the brain,
for drug discovery, and to model human brain diseases.
Other research teams have developed two- and
three-dimensional brain models with two to three cell types, but the latest
technique boasts a three-dimensional model of six cell types — neurons,
astrocytes, microglia, endothelial cells, pericytes, and oligodendrocytes. The
spheroids have all the qualities of BBB function: expression of tight
junctions, adherens junctions, adherens junction-associated proteins, and cell specific
markers. And the investigators have tested how different toxins can get through
when the model barrier is compromised.
The scientists said that the engineered tissue closely
resembles normal human brain anatomy, complete with neurons and immune cells.
They can use the model to study the effects of drugs once they cross the BBB,
and to identify small molecules that can reach specific brain tissue once it
gets through the semi-permeable barrier.
“The shortage of effective therapies (to treat brain diseases)
and low success rate of investigational drugs are due in part to the fact that
we do not have human-like tissue models for testing,” said senior author
Anthony Atala, MD, director of the Wake Forest Institute for Regenerative
Medicine. “The development of tissue engineered three-dimensional brain tissue
equivalents can help advance the science toward better treatments and improve
patients' lives.”
“This model is designed for a general understanding of any
compound going into the brain,” added Goodwell Nzou, a PhD candidate at Wake
Forest and co-author of the paper, published online in May in Scientific
Reports. “It can help us understand the effects of drugs that we want to use
for neurological conditions. Does the drug cross the BBB, and what does it do
to microglia, neurons, and oligodendrocytes? This kind of in-vitro system
allows us to ask and answer broad questions about the human brain.”
The scientists made induced pluripotent stem cell lines
(iPSCs) from human macrophage, human oligodendrocytes, and human neurons. Then,
they mixed them with cells that make up the neurovascular system — pericytes
and endothelial cells. They created a mini-neurovascular unit that they tested
to see whether it functioned as a model BBB. The endothelial cells and
pericytes coat the three-dimensional spheroids. Tight junctions form in the
barrier and the scientists can measure what goes in and what doesn't.
“First, we looked at the metabolic activity of individual
cell types to see whether the cells are viable,” explained Nzou. To do so, he
said they tagged cells with fluorescent proteins: green for alive and red for
dead. Around 85 percent of the cells in culture were still viable by day 21.
Then, they studied the major junctions found at the human BBB and tested their
permeability by adding a large protein — immunoglobulin G (IgG) — that normally
does not cross the barrier. In the mini-BBB wells they added histamine, which
is known to make the barrier leaky. As hoped, they saw that there was more IgG
moving through the barrier when histamine was added.
But what about small molecules? They used mercury chloride
to test whether the mini-BBB would stop the metal toxin from seeping through
the junctions. They used two models: the six-cell BBB and one made up of
neurons. As expected, there was abundant cell death when neurons was exposed to
mercury. But there was high metabolic activity in the wells filled with the six-cell
model, suggesting that it was keeping mercury out. All cell types were spared,
including the neurons.
They tested another toxin, MPTP
(1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine), implicated in Parkinson's
disease, which is known to cross the BBB and get taken up by astrocytes. They
coaxed the iPSC cells to become a population of dopamine neurons, and exposed
the spheroid to MPTP. They reported lower metabolic activity. The neuron model
escaped death from the toxin. (The scientists suspect that MPTP needs to be
metabolized to become toxic to neurons.)
Finally, they explored the hypoxic events that take place
during a stroke. Again, they did this in lab wells where they could control the
amount of oxygen going into the chamber. The hypoxic environment damaged the
spheroids and activated astrocytes.
“This model can tell us whether certain drugs prefer
specific cell types,” Nzou said.
Experts who were not involved with the research said the
study represents an advance in modeling six different cell types and could be
helpful for testing neurotoxicity as well as potential new drug therapies.
“Animal models have various limitations, as there are
species-to-species differences, and high-costs involved. They are labor
intensive, and there may be poor imaging accessibility for observations,” said
Takahisa Kanekiyo, MD, PhD, assistant professor in the department of
neuroscience at Mayo Clinic in Jacksonville, FL. “Current in vitro systems are
too simple to reconstruct the relevant physiological and pathological biology
in human brains. This novel 3D spheroid model is unique and interesting in terms
of reconstituting functional BBB with six main brain cell types,” Dr. Kanekiyo
said. “If future studies clarify the contribution of each cell type to BBB
integrity in the spheroid culture model, it might be more informative.”
Dr. Kanekiyo noted that since the BBB is dysregulated in
diverse neurological diseases, including Alzheimer's disease and stroke, the
BBB brain model would be useful in exploring disease pathogenesis. “Evaluating
the efflux of molecules from inside of the spheroid cultures is potentially
interesting to develop blood biomarkers for those diseases,” he said. “In
addition, it can be a useful tool to assess delivery of genes or drugs across
BBB, although the endothelial barrier integrity against smaller molecules needs
to be further elucidated.”
“Failures in recapitulating the human brain can result in
incorrect predictions of human toxicity and efficacy,” said Hansang Cho, PhD,
assistant professor in the department of mechanical engineering and engineering
science, biological sciences in the Center for Biomedical Engineering and
Science at University of North Carolina, Charlotte. “These investigators were
able to overcome these technical challenges, optimizing incompatible culturing
conditions and successfully co-culturing six different iPSC-derived and primary
brain cells for the first time. These in vitro models can demonstrate the
relevant functionality in human brains and may also become invaluable test-beds
for drug-discovery investigations and toxicology evaluations in human brains.”
https://journals.lww.com/neurotodayonline/Fulltext/2018/08020/At_the_Bench_Brain_Organoids__A_Novel_Brain_Model.2.aspx
Nzou G, Wicks RT, Wicks EE, et al Human cortex spheroid with
a functional blood brain barrier for high-throughput neurotoxicity screening
and disease modeling https://www.nature.com/articles/s41598-018-25603-5. Sci
Rep 2018: 8(1):7413
Abstract
The integral selectivity characteristic of the blood brain
barrier (BBB) limits therapeutic options for many neurologic diseases and
disorders. Currently, very little is known about the mechanisms that govern the
dynamic nature of the BBB. Recent reports have focused on the development and
application of human brain organoids developed from neuro-progenitor cells.
While these models provide an excellent platform to study the effects of
disease and genetic aberrances on brain development, they may not model the
microvasculature and BBB of the adult human cortex. To date, most in vitro BBB
models utilize endothelial cells, pericytes and astrocytes. We report a 3D
spheroid model of the BBB comprising all major cell types, including neurons,
microglia and oligodendrocytes, to recapitulate more closely normal human brain
tissue. Spheroids show expression of tight junctions, adherens junctions,
adherens junction-associated proteins and cell specific markers. Functional
assessment using MPTP, MPP+ and mercury chloride indicate charge selectivity
through the barrier. Junctional protein distribution was altered under hypoxic
conditions. Our spheroid model may have potential applications in drug
discovery, disease modeling, neurotoxicity and cytotoxicity testing.
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