Good Things Come in Small Packages: Neural Organoids’ Gift to Brain Science
Barely two weeks ago – in mid-April – STEMCELL Technologies of Vancouver, British Columbia, Canada, announced its new partnership with the Institute of Molecular Biotechnology (IMBA) in Vienna, Austria. Their collaboration will develop products for researchers who use “cerebral organoid cultures.” From a business standpoint, the international agreement sounds promising for both organizations. But what is an “organoid culture” anyway? And why are these little blobs of brain tissue so fascinating to researchers in science and medicine?
In late 2007, two independent research groups – in Japan and Wisconsin finally succeeded in deriving induced pluripotent stem cells (iPSCs) from adult human cells, thus avoiding the ethical issues associated with human embryonic stem cells. Through biochemical manipulation, the adult cells of origin could be “reprogrammed” to a more primitive state, then guided to develop into the desired cell type or lineage.
Before the work leading to their 2013 paper in Nature, IMBA scientists Madeline Lancaster and Jürgen Knoblich had cultured their neural stem cells developed from human iPSCs on flat (2-D) plates. But Lancaster, a postdoc in Knoblich’s lab, started noticing that some cells tended to form 3-D clumps and “fall off the plate.” With a touch of the same intuition and curiosity that kept Alexander Fleming from trashing his moldy Staphylococcus culture, Lancaster looked at the little clumps and thought: “These are cool – let’s see what happens if I let them keep growing”
Madeline Lancaster | Picture: EMBL
Jürgen Knoblich | Picture: IMBA
Brains in a Bowl?
Despite her commitment to salvage the clumps of cells, the cultures faced a very fundamental challenge: With no blood supply, their innermost cells were starved for nutrients and oxygen. Lancaster enclosed each clump in a nurturing structural protein matrix (Matrigel), then transferred a number of clumps to liquid culture medium in a spinning bioreactor, constantly rinsing medium over the organoids to enhance nutrient absorption. Within 20-30 days, the clumps began to show discrete brain regions and within two months they achieved their maximum size, usually surrounding a fluid-filled cavity much like neural stem cells around the ventricles of an intact brain. The challenge of survival was solved, but nutrient access still limited the size of organoids to 4 mm or less.
Neural stem cells (magenta) and neurons (green) comprise brain organdies. Lancaster’s group has nurtured organoids/clumps like this one these for up to 15 months | Photo The-Scientist.com
Organoids make no claim to becoming replacement organs. The suffix “-oid” means “resembling” or “like” – as in android, ovoid, or factoid. Not only are organoids limited in size, but the knowledge and technology needed to develop fully functional brains (or hearts or kidneys) lie far in the future. What organoids do offer, however, is an organized group of defined human cells that are fully compatible with the immune system of their iPSC source. Even more importantly, they express the source’s genome and biochemical pathways.
Once the survival of normal human neural organoids was ensured, the next question was: Can they be used to study human neuropathology? “Organoids are poised to make a major impact on the understanding of disease, and also human development,” said Arnold Kriegstein, a brain scientist at the University of California, San Francisco. That would mark a significant departure from the long-established pattern of using animal models to study human disease.
Microcephaly: Producing Neurons Too Early
Lancaster, now at the Medical Research Council Laboratory of Molecular Biology in Cambridge, England, sometimes compares the neural organoids in her laboratory to an airplane with oddly placed parts. “It can’t actually fly … but you can study each of the components individually and learn lot about them.”
Her research on microcephaly, a congenital human disorder in which the developing brain produces too few neurons, began at IMBA and continues in her present position. Microcephaly is externally evident in the patients’ abnormally small heads (thus the name of the disease). No equivalent syndrome exists in mice, even when their genomes are manipulated to produce the same mutation, so mouse models were never a good route to solving this neurological problem.
Most often, microcephaly has a genetic cause (a homozygous mutation in the microcephalin gene), but in some cases the problem is attributed to early closure of the cranial sutures, pregnancy complications, drug, alcohol, or toxin exposure during pregnancy, malnutrition, et al. Recently, microcephaly has made the headlines because of its association with Zika-virus infections in pregnant women. Although microcephalic children can have normal intelligence, the occurrence of mental retardation, developmental delays, seizures, and balance problems is quite high.
How does all this relate to neural organoids? Primarily because Lancaster and Knoblich, and now other researchers as well, have used neural organoids to decipher the developmental processes underlying the downsized brain and cranium.
Taking skin fibroblast cells from individuals with microcephaly, they reprogrammed the cells into iPSCs, cultured them to form neural organoids, and noted that these organoids were abnormally small and stunted -- like the brains of microcephalic patients. Pursuing this further, Lancaster and Knoblich discovered that their “microcephalic” organoids contain fewer neural progenitor cells, fewer glial stem cells, but more neurons than organoids derived from normal human skin cells! Apparently, rather than replenishing themselves during early stages of growth, the abnormal stem cells start producing neurons too early – running out of steam before a full neuronal population can be generated. Their collaboration with Andrew Jackson at the University of Edinburgh also found that replacing the defective protein in microcephalic organoid cultures can partially normalize the organoids’ growth.
Generation of 3D Endodermal and Ectodermal Organoids from Skin Fibroblasts
Reprogramming of fibroblasts into induced pluripotent stem cells (iPSCs) enables in vitro differentiation into tissue-specific progenitors, such as intestinal stem cells, thyroid, or neuroectodermal progenitors. When grown under the right conditions in suspension, these progenitors self-organize into 3D tissues resembling the intestine, thyroid, optic cup, or cerebral cortex. | Image: Cell
ASD: Inhibitory Neurons Tip the Balance
Another research application of neural organoid cultures – and an example of how rapidly the new technology has spread since its 2013 beginnings at IMBA – focuses on autism spectrum disorder (ASD). ASD individuals display a broad spectrum of traits including impaired social development and communication, repetitive behaviors, unusual responses to sensory stimuli, atypical eating behavior, and sleep problems. Even though patients with idiopathic autism do not appear to share a single genetic cause, their cells exhibit similar types of dysregulation.
Flora Vaccarino, a leading researcher in the Child Study Center at Yale School of Medicine, recognized the potential of iPSC for investigating causes of ASD. “I immediately realized that this could be used to reenact stages of neuro-development that were almost impossible to study in humans,” she said.
Her team obtained skin cells from four male adolescents with ASD and from their respective fathers, none of whom has ASD. After the cells were reprogrammed into iPSCs, they were biochemically guided to differentiate into neural organoids resembling embryonic precursors of the cerebrum – specifically, the mid-fetal stage of cortical development. Unlike microcephalic organoids, ASD organoids exhibit no gross morphological differences. But ASD and “neuro-typical” cells differed in terms of their gene expression: ASD organoids were likely to show increased activity of genes for cell proliferation, neuronal development, and synapse assembly.
“The cells of the patients divide faster than the fathers’,” said Vaccarino. They then investigated whether all the cells, or just certain types, were dividing faster. Interestingly, the number of excitatory neurons was unaffected, but the number of inhibitory neurons was increasing faster than normal. Genetic analysis revealed the upregulation of messenger RNA encoding FOXG1, a protein important in brain development. By blocking this factor in organoids derived from ASD patients’ cells, the normal balance between excitatory and inhibitory neurons was restored.
“We hope to use the same system to investigate other genes that may be involved in ASD,” said Vaccarino. Her group has already found that expression of FOXG1 – and other genes differently expressed in ASD individuals – correlates with greater head circumference and with the severity of autism symptoms.
Reason for Optimism
It’s hard to overstate the potential of neural organoids for investigating human neuropathology. Diseases proposed for future (and present) studies by groups around the world include Alzheimer’s disease, schizophrenia, amyotrophic lateral sclerosis (ALS), and Parkinson’s disease, as well as military research into traumatic brain injury and post-traumatic stress disorder (PTSD). Small wonder that STEMCELL Technologies was eager to establish that licensing agreement with IMBA, which will enable them, exclusively, to develop products for international researchers wanting to undertake the culture of cerebral organoids.
“With this technology, there finally exists an in vitro culture system that gives us a glimpse into the complexities of the human brain, undoubtedly improving our understanding of brain development and neurological disease,” said Allen Eaves, CEO and President of STEMCELL Technologies.
Jürgen Knoblich, at IMBA, is equally enthusiastic about the agreement: “We … hope that by making this technology available, we’ll enable scientists from across the globe to develop other models for neurological diseases and disorders.”
Given the past accomplishments of their respective labs and institutions, both parties to this international agreement have good reason to be optimistic. And so do a huge population of individuals throughout the world, watching hopefully as scientists use this exciting new research technology to pursue the causes, preventions, and therapies for their neural diseases.