Lab-grown model 3D brains

"Cerebral organoids” can model complex human brain disorders and the earliest stages of brain development
August 30, 2013

Cross-section of cerebral organoid. Neural stem cells in red and neurons in green. (Credit: M. A. Lancaster et al./Nature)

Scientists in an Austrian laboratory have developed complex human brain tissue made from stem cells in a laboratory 3D culture system for the first time. The method allows induced pluripotent stem (iPS) cells (which have the potential to differentiate into almost any cell in the body) to develop into “cerebral organoids” — or “mini brains.”

These mini brains, which are a few millimeters across, develop several discrete but interdependent brain regions, including a cerebral cortex containing progenitor populations that organize and produce mature cortical neuron subtypes.

Studies of the human brain’s development and associated human disorders are extremely difficult, as no scientist has thus far successfully established a three-dimensional culture model of the developing brain as a whole. The complexity of the human brain has also made it difficult to study many brain disorders in model organisms, highlighting the need for an in vitro (lab) model of human brain development.

How to build a mini-brain

The scientists at the Institute of Molecular Biotechnology (IMBA) of the Austrian Academy of Sciences (OeAW), led by Dr. Jürgen Knoblich, fine-tuned growth conditions and provided a conducive environment.

Cues from the stem cells guided the development towards different interdependent brain tissues. The scientists were also able to model the development of a human neuronal disorder and identify its origin.

Cerebral organoid culture system. Example images of each stage are shown. bFGF, basic fibroblast growth factor; hES, human embryonic stem cell; hPSCs, human pluripotent stem cells; RA, retinoic acid. (Credit: M. A. Lancaster et al./Nature)

Starting with established human embryonic stem cell lines and iPS cells, IMBA scientists identified growth conditions that aided the differentiation of the stem cells into several brain tissues. They used media for neuronal induction and differentiation to avoid the use of patterning growth factor conditions, which are usually applied to generate specific cell identities from stem cells.

Knoblich explains the new method: “We modified an established approach to generate neuroectoderm, a cell layer from which the nervous system derives. 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. In order to enhance nutrient absorption, we later transferred the gel droplets to a spinning bioreactor. Within three to four weeks defined brain regions were formed.”

Spinning bioreactor system (credit: IMBA)

After 15–20 days, “cerebral organoids” formed that consisted of continuous tissue (neuroepithelia) surrounding a fluid-filled cavity reminiscent of a cerebral ventricle.

After 20–30 days, defined brain regions, including a cerebral cortex, retina, meninges as well as choroid plexus, developed. A

After two months, the mini brains reached a maximum size, but they could survive indefinitely (currently up to 10 months) in the spinning bioreactor.

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.

Modeling microcephaly in mini brains

The new method also offers potential for establishing model systems for human brain disorders. These models are urgently needed because the commonly used animal models are of considerably lower complexity, and often do not adequately recapitulate the human disease.

Knoblich’s group has demonstrated that the mini brains offer great potential as a human model system by analyzing the onset of microcephaly, a human genetic disorder in which brain size is significantly reduced. By generating iPS cells from skin tissue of a microcephaly patient, the scientists were able to grow mini brains affected by this disorder.

As expected, the patient-derived organoids grew to a lesser size. Further analysis led to a surprising finding: while the neuroepithilial tissue was smaller than in mini brains unaffected by the disorder, increased neuronal outgrowth could be observed.

This 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 that 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 cause the disorder.

“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,” explains Dr. Madeline A. Lancaster, team member and first author of the publication. “They allow for the testing of therapies against brain defects and other neuronal disorders. Furthermore, they will enable the analysis of the effects that specific chemicals have on brain development.”

According to The Scientist, Knoblich cautioned that the organoids are not “brains-in-a-jar.” “We’re talking about the very first steps of embryonic brain development, like in the first nine weeks of pregnancy,” he said. “They’re nowhere near an adult human brain and they don’t form anything that resembles a neuronal network.” These models will not help to unpick the brain’s connectivity or higher mental functions, but they are excellent tools for studying both its early development and disorders that perturb those first steps.