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.”
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
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.