Creamy white spheres, roughly the size of peas, immersed in a swirling pink solution. To the untrained eye, cerebral organoids are deceptively unimpressive. However, the immense potential they hold for unlocking the secrets of the human brain is, indeed, nothing to be balked at. These self-assembled, laboratory-grown tissues are continually pushing the boundaries of neurodevelopmental and neurodegenerative research. Recent advancements in neural tissue engineering are creating a buzz in the scientific community: are bigger and better “mini-brains” in store?

The iPSC toolkit for neuroscience

Before organoids, neuroscientists were predominantly reliant upon animal models, post-mortem human brain samples and 2D neural cell cultures. However, these represent relatively rudimentary techniques, given the sheer complexity of the brain’s anatomy: a hundred billion neurons and glial cells arranged in an intricate and patterned circuitry, with a highly dynamic genetic and biochemical profile.

In 2006, a landmark discovery by Nobel prize winner Shinya Yamanaka triggered a major paradigm shift in brain analysis methodologies. He found that the introduction of specific genes to mature adult cells – a process called “reprogramming” – reverted them to an embryonic stem cell-like state. The resulting induced pluripotent stem cells (iPSC) came with the ability to not only divide infinitely, but given the right environmental conditions, the capacity to differentiate into almost any cell type in the human body, including neurons. Protocols for coaxing iPSCs into radial formations of neural progenitor cells, known as rosettes, were subsequently devised. This provided researchers with a platform from which human neural cells of various lineages could be generated.

Discovery and innovation in organoid architecture

From here, neuronal cultures gradually transitioned from flat cellular monolayers into the third dimension. In 2013, Postdoctoral Fellow in Jürgen Knoblich’s laboratory (IMBA, Vienna), Madeline Lancaster, made a deft observation in her neural progenitor cell cultures. During their growth and maturation, some cells sometimes spontaneously formed spheroidal clusters, which were suspended above their plastic-bound counterparts. Out of curiosity, Lancaster collected these cellular agglomerations and maintained them in a synthetic gel-based medium, which closely resembled the neuronal microenvironment. Cross-sections of these tissues revealed a spectacular phenomenon: these neurons had self-organized in a spatially-defined manner, forming many hallmark characteristics of the developing brain.

Excitingly, neurons within these tiny structures not only mirrored anatomical aspects, but were also found to be physiologically functional. Sergiu Pasca’s group at Stanford University found that almost 90 percent of cortical neurons within these spheroids fired electrical impulses through active synapses. Organoid slices subjected to electrophysiological recordings are able to offer never before seen insights into how and why neural circuits go awry in the case of certain neuropsychiatric disorders.

Scientists are also harnessing the power of mini-brains to bridge other important knowledge gaps. Neurodevelopmental biologist Andrew Jackson (University of Edinburgh) effectively leveraged cortical organoid technology to identify a key protein, CDK5RAP2,  involved in the onset of the neurodevelopmental disorder, microcephaly. Fascinatingly, Jackson and his team were able to partially restore a normal phenotype in these microcephaly patient-derived organoids by replacing the faulty protein. Similar pathways have been elucidated in a growing list of neurodevelopmental diseases: autism, schizophrenia and epilepsy, to name a few.

Organoids 2.0: breaking barriers

Despite their many promises, though, one of the major pitfalls of organoids is that not all brain cell types are adequately represented within them. Oligodendrocytes, microglia, interneurons and inhibitory neurons, for instance, are generally lacking. From a developmental perspective, the ventricular and subventricular brain regions (established early during embryogenesis) were found to be distinctly present in organoid cross-sections. After two months in culture, however, an upper limit was reached, in which they only bore transcriptomic resemblance to brains at the second trimester of life.

The absence of a blood supply presents a major hurdle that impedes further cortical organoid maturation. To a limited extent, this can be counteracted by suspending organoids in spinning bioreactors and cultivating them for periods from months to years. In this way, a continuous and dynamic flow of crucial nutrient resources and the removal of cellular waste products is created.

Nonetheless, significant restrictions to the final organoid size and a high degree of variability still persist without a capillary framework – until now. Neurosurgeon, Ben Waldau (UC Davis Medical Center), recently reported that organoids have finally received this long-awaited upgrade. For the first time, iPSCs established from a single patient were simultaneously differentiated into both vascular cells and neural cells, maintained together in a single organoid. Surprisingly, after three to five weeks, an impressive web of capillaries had formed, prominent mostly around the circumference of the organoid, with some penetrating into the deeper internal regions. By supplying oxygen and nutrients to a larger proportion of neurons, this vascular meshwork can allow organoids to grow larger and conceivably, more anatomically complex.

Waldau and his team also found that his vascularized organoids survived a drastic change of scene, from a dish, into a living brain. In a concurrent experiment, he tested their robustness in an in vivo setting, by implanting them into the cranium of a mouse. After two weeks, the human brain organoids were found not only to be thriving in the rodent’s brain, but had also amassed even more blood vessels. Clinically, this would be revolutionary. Neural damage from stroke and injuries, for example, could potentially be repaired using vascularized organoid tissues straight from the patient.

New horizons for neural organoids

For now, a true miniaturized laboratory version of the human brain remains in the realm of science fiction. Nevertheless, the neural organoid evolution continues; the aim is to create increasingly refined, reproducible and scalable brain models. Multidisciplinary efforts to achieve this, from 3D printed bioreactors and microfluidic-based “brain-chips” to gene editing technologies like CRISPR/Cas9, mean organoids’ next critical upgrade is surely near at hand.

References

  1.  Dishing out mini-brains: Current progress and future prospects in brain organoid research
  2. Induction of Pluripotent Stem Cells from Adult Human Fibroblasts by Defined Factors
  3.  Directed differentiation of human pluripotent stem cells to cerebral cortex neurons and neural networks
  4.  Generation of cerebral organoids from human pluripotent stem cells.
  5.  Organogenesis in a dish: Modeling development and disease using organoid technologies
  6. Functional cortical neurons and astrocytes from human pluripotent stem cells in 3D culture
  7.  Assembly of functionally integrated human forebrain spheroids
  8.  Cerebral organoids model human brain development and microcephaly
  9.  Psychiatry in a Dish: Stem Cells and Brain Organoids Modeling Autism Spectrum Disorders
  10.  Cerebral organoids reveal early cortical maldevelopment in schizophrenia—computational anatomy and genomics, role of FGFR1
  11.  Using Patient-Derived Induced Pluripotent Stem Cells to Model and Treat Epilepsies
  12.  Self-organized cerebral organoids with human specific features predict effective drugs to combat Zika virus infection
  13.  Cerebral organoids model human brain development and microcephaly
  14.  Cell diversity and network dynamics in photosensitive human brain organoids
  15.  Generation of human vascularized brain organoids
  16.  Brain Region-specific Organoids using Mini-bioreactors for Modeling ZIKV Exposure
  17.  Microfluidic organ-on-chip technology for blood-brain barrier research
  18.  Organoid technologies meet genome engineering

Tara Fernandez


Tara Fernandez
is a Vancouver-based Cell Biologist and science communicator. Her research focuses on new ways to treat cells on the nanoscale.