A cross-section of an entire organoid showing development of different brain regions. All cells are in blue, neural stem cells in red, and neurons in green. [© IMBA/Madeline A. Lancaster]
Researchers from the Institute of Molecular Biotechnology (IMBA) of the Austrian Academy of Sciences report the development of human brain tissue in a 3D cell culture system. Their technique, which is discussed in an article in the current issue of Nature (“Cerebral organoids model human brain development and microcephaly”), permits pluripotent stem cells to develop into cerebral organoids, or “mini brains,” that consist of several discrete brain regions.
The scientists were able to see the dorsal cortex throughout the cultures, the ventral cortex in one-third of cultures, and the retina in about ten percent of their cultures. They rarely saw the hippocampus and never viewed the cerebellum.
“We modified an established approach to generate so-called neuroectoderm, a cell layer from which the nervous system derives,” explained Jürgen Knoblich, Ph.D., IMBA’s deputy scientific director, during a press conference. “Fragments of this tissue were then maintained in a 3D culture and embedded in droplets of a specific gel that provided a scaffold for complex tissue growth. To enhance nutrient absorption, we later transferred the gel droplets to a spinning bioreactor. Within three to four weeks defined brain regions were formed.
“We want to create a model system of the human brain. [However,] we optimized our cultures to model the dorsal human cortex. This allows us to study the early steps of human brain development.”
“In the dorsal cortical regions of the organoids, stem cells look like those in a human embryo at nine or ten weeks,” added Madeline Lancaster, Ph.D., who works as a Marie Curie postdoctoral fellow at the IMBA. She pointed out that although the organoids contained all the brain regions present in a normal human embryo at nine weeks, they are not spatially organized.
How to Build a Mini Brain
According to Dr. Knoblich, the researchers began their work with established human embryonic stem cell lines and then induced microcephalic patient-derived cells to turn into pluripotent stem (iPS) cells to generate organoids. “We wanted to compare the organoids to healthy cells,” he said.
Intrinsic cues from the stem cells guided the development toward different interdependent brain tissues. Using the mini brains, the scientists were able to model the development of a human neuronal disorder (i.e., microcephaly) and identify its origin.
“A normal developing brain has a stem cell population that undergoes rounds of division at specific times to make more stem cells and eventually neurons,” noted Dr. Lancaster. “But the microcephalic patient-derived stem cells made neurons too early in the process. This led to a depletion of the stem cell population, which resulted in fewer neurons being made.”
Putting it another way, Dr. Knoblich explained that this finding led to the hypothesis that, during brain development of patients with microcephaly, the neural differentiation happens prematurely at the expense of stem and progenitor cells, which would otherwise contribute to a more pronounced growth in brain size. “Further experiments also revealed that a change in the direction in which the stem cells divide might be causal for the disorder,” he continued.
The mini brains had reached their maximum size after two months, but they could survive indefinitely (currently up to 10 months) in the spinning bioreactor, according to the scientists. Further growth, however, was not achieved, most likely due to the lack of a circulation system and hence a lack of nutrients and oxygen at the core of the mini brains. “In normal brain cells the vasculature brings in essential nutrients,” explained Dr. Knoblich.
Potential Future Uses
The new method offers great potential for establishing model systems for human brain disorders, according to the researchers. Such models are urgently needed, as the commonly used animal models are of considerably lower complexity and often do not adequately recapitulate the human disease.
In addition to the potential for new insights into the development of human brain disorders, mini brains will also be of great interest to the pharmaceutical and chemical industry, said Dr. Lancaster.
“They allow for the testing of therapies against brain defects and other neuronal disorders,” she explained. “Furthermore, they will enable the analysis of the effects that specific chemicals have on brain development.”
When asked if organoids could eventually be used to grow replacement parts for the human brain, Dr. Knoblich expressed pessimism.
“The ultimate complexity of the brain does not allow for replacement of structures. This is the case because the different parts of an adult brain are so tightly integrated,” he explained.
But regarding new ways to treat diseases like schizophrenia and autism, Dr. Knoblich sounded a more positive note while acknowledging that such therapies based on the team’s current research are way down the line.
“Disorders like schizophrenia and autism typically manifest themselves during adulthood,” he said. “But the defects themselves take place during development. We want to model these diseases in the future and see if we can get a better understanding of these early events.”