Abstract
Inspired by the iron-clad tenacity of past scientists we set out to understand the mechanisms of life’s bricks, how they divide, which genes direct them, how they differentiate into specific lineages and how this intricate orchestration goes awry.
Much of the bridging between past and contemporary neuroscience lies in maintaining a close-knit relationship between the sciences that study the mind and reductionist science. It is through health that we conceptualize the fundamental properties of its nature but through disease that we appreciate them. Neurological disorders gather our attention as they challenge and alter communication and relationships by limiting and then extending them. To study phenotypes associated with human brain development, function, and disease, it is necessary to use experimental systems that are accessible, ethically justified, and can replicate human context.
Human pluripotent stem cell (hPSC)-derived brain organoids offer such a system, which faithfully reiterates features of early human neurodevelopment in vitro, including the generation, proliferation, and differentiation of neural progenitors into neurons and glial cells and the complex interactions among the diverse, emergent cell types of the developing brain in three-dimensions (3D). In recent years, numerous brain organoid protocols and related techniques have been developed to recapitulate aspects of embryonic and fetal brain development in a reproducible and predictable manner. Coupling our ground-breaking cerebral organoid technology with elegantly tailored cutting-edge genetic manipulations has enabled us to efficiently screen for disease-linked mutations serving as a boon when studying human neurobiology and neurodevelopmental disorders. Altogether, these different organoid approaches provide distinct bioassays to unravel novel, disease-associated phenotypes and mechanisms.
To say that the human cerebral cortex is the organ of civilization is to lay a very heavy burden on so small a mass of matter. — C. Judson Herrick (1926)
Understanding the brain is probably the greatest task of human biology for it encapsulates the purpose of humankind. The brain births ideas, feelings, cultures, motion, reflex, wisdom, and identity. It is an organ that has nothing short of divided and fused, perplexed and intrigued populations throughout history. The light shines on it; no dimming in sight, not even so much as a flicker as we gather ourselves sedulously attempting to satisfy our scorching thirst from ploughing away its many facets for the chance of grasping its veritable nature.
The Greek philosopher Aristotle believed that memory and consciousness were found in the heart, coining what we now refer to as emotional intelligence. The Egyptians, however, first described the basic anatomy of the brain and made the connection that it controls movement. The brain as an organ has interested populations and cultures amass, many reaching the same conclusions. Much of the early knowledge was based on observations by doctors who made poignant connections between human behavior, physiology and the brain.
A thousand years later the French philosopher Descartes distinguished the brain from the mind, thereby introducing the pertinent notion of dualism. This planted the seed that flowered into the scientific revolution where the brain earns the leading role sprouting different fields of neuroscience research. Like all sciences, neuroscience was approached from the macroscopic to the microscopic scale, offering plenty of fascinating discoveries and contributing to deep and often controversial discussions along the way.
The fascination over the complexity and importance of the human brain ensures quick progress in teasing out its many layers of anatomy and diverse functions. Strolling along the neuroscience history aisles one notices the quick succession of discoveries with Purkinje (Purkyně) describing the neuron, Broca identifying the region responsible for speech and Pavlov examining the physiology of involuntary reflexes earning him a Nobel Prize in Medicine. Soon after, electroencephalography (EEG) is developed to measure electrical activity in the brain and Sir Charles Sherrington wins the Nobel Prize for describing the existence of synapses and how reflexes occur as a result of nerves extending into muscles. A decade later Isidor Rabi wins the Nobel Prize for discovering nuclear magnetic resonance which made the development of magnetic resonance imaging (MRI) possible. Several researchers bring evidence that solidified Julius Bernstein’s hypothesis that action potential is a product of ionic conductance. These technological breakthroughs permit Joseph Erlanger and Herbert Gasser to document the existence of different action potentials across different cells which ultimately leads to their Nobel Prize-winning discovery of the velocity of action potentials. Progress in physiological neuroscience is accompanied by the confirmation that acetylocholine is a neurotransmitter marking a landmark discovery for molecular neuroscience.
The variety of emerging fields quickly creates the need for more unified efforts in teasing out the complexity of our brain and it is now officially recognized as an independent discipline. The fast-paced progress in its diverse fields highlights the need for a deeper understanding of our nervous system by looking down to its founding units.
At this point a set of groundbreaking discoveries are made that influence and impact biology and medicine globally in an unprecedented way. Wilhelm His and Santiago Ramón y Cajal independently notice the presence of cells from which all types of neurons arise before migrating from the place of origin to increasingly more distant locations.1,2 Ernest McCulloch and James Till identify the existence of cells in the adult bone marrow which can self-renew and are hematopoietic giving rise to all blood cell types, inaugurating the field of stem cell research.3 As scientists curiously peak down the microscope into life’s building blocks, cell biology and medicine have, unbeknown to them, been revolutionized. Martin Evans and Matthew Kaufman isolate and culture mouse embryonic stem cells opening up the possibility to study the function of specific genes during disease.4 Soon after, James Thompson reports the derivation and culturing of human embryonic stem cells that retain their pluripotent state; their ability to give rise to different cell types.5 Shinya Yamanaka astounds the scientific community by making a remarkable discovery that adult mouse fibroblasts can be reprogrammed into reacquiring a pluripotent state, much like that of mouse embryonic stem cells and calls them induced pluripotent cells (iPSCs).6 Together with Takahashi they successfully derive iPSCs from human fibroblasts as well, alleviating the considerable and understandable ethical concerns of using human embryonic material for stem cell research.7
The hallmark properties of stem cells are the ability to self-renew by dividing indefinitely into daughter cells, while at the same time retaining the capacity to commit daughter cells to lineage-specific differentiation which is the more differentiated progeny that drives tissue specific development. Stem cells can be isolated from the blastocyst stage of the developing embryo, but they are also found to persist in niches of adult tissues, including the brain. Neural stem cells, like all stem cells, play important roles in tissues homeostasis, and in development. In adult organisms, they ensure continuous replacement of dying or damaged cells, while during development they generate most of the cell types in a developing organ. To fulfill this task, stem cells can maintain an undifferentiated state, but at the same time generate daughter cells that are lineage-restricted and ultimately undergo terminal differentiation. Understanding how the balance between self-renewal and differentiation is controlled within a stem cell lineage is important since defects in the control of this process can result in tissue degeneration or tumorigenesis. Neural stem cells are the focus of my lab’s research, and our work has offered many insights into what is “there”.
Building on the fundamental idea that biological mechanisms are conserved throughout evolution, biomedical research focuses on animal model organisms. Animal experimentation is widely used as a proxy for understanding human embryonic development and organ function.8 A menagerie of animal species, both vertebrate and invertebrate, are employed in an attempt to answer more direct questions. Each model offers particular strengths (Fig 1).9 Although some extrapolations lead to valid knowledge, other speculations do not translate quite as fluently. Human physiology is profoundly different from the mouse model system: it is perhaps unsurprising that there are huge differences in metabolism between humans and laboratory models, given that humans develop far slower than the other models10 or the fact that continuous oscillations in the hippocampus, for the purpose of spatial navigation of rodents, are found not to be true in bats or monkeys. Even further, several biological phenomena that are specific to humans are not amenable to being reproduced in animal models. The human brain, for example, is far more complex than its mouse counterpart, owing partly to human-specific developmental events and mechanisms.11 Neurons in the human cortex, for example, arise from a cell type (outer radial glia) that is either not present — or is present only in minute numbers — in rodents.11 Despite this and with, perhaps, a reluctant recognition that not all knowledge from the animal kingdom transcribes to the human, armies of scientists methodically reveal distinct aspects of brain development by using animal models.
Drosophila and Caenorabditis elegans models are instrumental in elucidating the principles of stem cell self-renewal and differentiation, uncovering molecular parallels for this process in different species. Understanding that one way to generate cellular diversity during development is to segregate cell-fate determinants predominantly into one daughter cell upon division, inspires us and others to ask how this process comes to be. Work mostly done in the fruitfly, Drosophila, suggests two different mechanisms by which this remarkable task can be achieved.12 Already in interphase, cells which undergo such intrinsically asymmetric divisions use apical-basal or planar polarity of the surrounding tissue to set up an axis of polarity. As they enter mitosis, this axis is used to polarize the distribution of protein determinants and to orient the mitotic spindle so that these determinants are inherited by only one of the two daughter cells. Alternatively, they can orient their division plane so that only one of the two daughter cells maintains contact with the niche and stem cell identity (Fig. 2).13 A stem cell, by orienting its mitotic spindle perpendicularly to the niche surface, ensures that only one daughter cell can maintain contact with the stem cell niche and retain the ability to self-renew. In contrast to intrinsically asymmetric cell divisions, which usually follow a predefined developmental program, niche-controlled stem cell divisions offer a high degree of flexibility. Occasionally, the stem cell can divide parallel to the niche, thereby generating two stem cells to increase stem cell number or to compensate for occasional stem cell loss. For this reason, niche mechanisms are more common in adult stem cells, whereas intrinsically asymmetric divisions predominate during development.
Clarifying the mechanism of asymmetric cell division in the Drosophila nervous system becomes the starting point of my contributions to stem cell biology. Building on what I learned during my post-doctoral work,14,15 I team up with extraordinary and brave scientists in my lab to develop a conceptual framework for how the asymmetric cell division process occurs. We propose, test, and show that an axis of polarity is established during interphase guiding both the orientation of the mitotic spindle and the asymmetric localization of protein determinants during mitosis. Over several years, we identify a near-complete set of proteins involved in the various stages of the process and achieve a mechanistic understanding of asymmetric cell division. We find that it is the asymmetric localization of the so-called Par-proteins that establishes the polarity axis to guide asymmetric cell division. In mitosis, a polarized attachment site for microtubules established by the proteins Pins, Galphai and Mud orients the mitotic spindle while the kinase aPKC detaches protein determinants (Numb, Prospero and Brat) from one side and guides their accumulation at the opposite site16–20 (Fig. 3. This mechanism enjoys wide acceptance in the field and becomes part of most developmental biology textbooks. Importantly it is conserved in mammalian stem cells highlighting the relevance of asymmetric cell division in stem cell biology especially considering the compelling connections to tumorigenesis that begin to emerge. Like, for example, the link we make between cellular metabolism and immortalization of tumor-initiating cells by performing targeted metabolomics and in vivo genetic screening.21
Matching a gene to its function is necessary in detangling developmental processes but it is also a laborious process. Genetic screens become the go-to method for the elucidation of developmental pathways and work done in invertebrates is followed by an analysis of evolutionary conservation in mammalian model systems, often leading to clinical translation for humans. Pioneers, Christiane Nüsslein-Volhard and Eric Wieschaus pave the way by screening through massive numbers of randomly induced mutant fly embryos for defects in developmental patterning and classify 15 genes as the key players during embryonic development in Drosophila.22 In the wake of the discovery of stem cells and the establishment of the key tenets of stemness, another technology is appearing in an entirely unexpected way. As Andrew Fire and Craig Mello investigate gene expression regulation in C. elegans, they observe that double-stranded RNA blocks the expression of the respective gene and name this approach RNA interference (RNAi).23 The ability to silence specific genes overcomes the main drawback of random mutagenesis approaches in that it is gene specific. RNAi allows large-scale genetic screens to reveal the functions of many genes through development. Consequently genome-wide RNAi studies are performed in mammalian stem cell cultures.24,25
Naturally, the wish to study stem cells in situ arises, where the interactions with the surrounding niche and the tissue-specific characteristics of individual lineages are maintained. In Drosophila, this becomes possible through the establishment of a transgenic RNAi library that can be expressed in a tissue-specific manner.26 Together with my team we become the first to perform genetic screens in a tissue-specific manner within an entire organism. We focused on external sensory organs, where defects in asymmetric cell division or Notch signaling lead to visible phenotypes, like gain or loss of bristles. We use a library of 20,000 transgenic RNAi lines generated by Barry Dickson that result in informative loss-of-function phenotype data for 23% of all protein coding Drosophila genes, a data set that is still regularly queried by others in the field.27
Armed with all this knowledge, we now wonder how the finely tuned yet fragile homeostatic balance between stem cell self-renewal and differentiation is regulated. We perform genetic screens on neural stem cells using genome-wide transgenic RNAi and identify 620 genes that are potentially involved in controlling this balance in Drosophila neuroblasts (larval brain stem cells).28,29 We quantify all phenotypes and derive measurements for proliferation, lineage, cell size, and cell shape. We identify a set of transcriptional regulators essential for self-renewal and integrate hierarchical clustering with interaction data to create functional networks to uncover the control of neuroblast self-renewal and differentiation. Our data reveal key roles for the chromatin remodeling Brm complex, the spliceosome, and the TRiC/CCT-complex showing that the alternatively spliced transcription factor Lola and the transcriptional elongation factors Ssrp and Barc control self-renewal in neuroblast lineages.28,29 These efforts truly lay solid foundations for the mechanistic discoveries that ensued on stem cell immortalization and tumorigenesis.
Studies in Drosophila, undoubtedly enriched our scientific acumen of neural development but the gnawing need to intimately explore the least understood organ of our body, is ever-present. The complex architecture and function of the human brain enables us to perform higher cognitive functions. Abnormalities in the structure or function of the brain can lead to severe neurological and psychiatric disorders. It is becoming increasingly clear that many neurological and psychiatric disorders have their roots in neurodevelopment.30,31 However, determining the neurodevelopmental cause and mechanisms of these brain disorders is challenging, due to the limited access to the human brain tissues. Given the large evolutionary distance between mouse and human, and the immense elaboration of the primate brain in size and complexity, there are many features unique to human brain development and diseases that are not seen in rodent systems.32
Standing on the shoulders of giants we can now see much farther than we ever thought possible and diving into the unknowns of the human brain seems to be more within our reach. But first a bridge must be built. A new model that does not rely on human primary material is needed. Madeline Lancaster in my laboratory, replaces mortar and pestle with pipette and culture dish and attempts to use human pluripotent stem cells to model key developmental events of the human brain in vitro. By combining classical cell culture approaches with recently developed methods enabling cells to grow three-dimensionally, we develop cerebral organoids, a tissue culture method that recapitulates human brain development.33 The gap between animal models and human beings has been bridged.
Human brain organoids, otherwise known as cerebral organoids, are hPSC-derived self-organizing human pluripotent stem cell-derived three-dimensional culture systems that develop various discrete, although interdependent, brain regions. Cerebral organoids recapitulate the neurodevelopmental scheme to generate 3D tissue architectures that mimic various features of the developing fetal brain pertaining to cellular composition and tissue structure.34 hPSCs cultured in appropriate media conditions form an embryoid body35 or a spheroid36 and undergo neural induction to adopt the neuroectodermal fate.33,36,37 The neuroectodermal progenitors self-organize into multiple 3D structures featuring apical lumens called neural rosettes or neural buds reminiscent of the neural tube. After 1 month in culture, organoids exhibit neuronal differentiation (Tuj1, Fig. 4), leading to progressive expansion and thickening of cerebral tissues (Fig. 4).38 By 2 months, different brain regions are visible, including forebrain and hippocampus (Fig. 5).38
The path to cerebral organoid generation is exciting but nothing short of challenging. Knowing we have made the first big leap into the systematic investigation of human brain development and disease we dedicate much time and effort to deepen our understanding and broaden our tool kit. We use brain organoids to examine the cell biological basis of a form of microcephaly, a disorder involving small brain size.33,39,40 In fact, a variety of neurological disorders can be examined in cerebral organoids. We use RNA interference and patient-specific induced pluripotent stem cells to model microcephaly, a disorder that has been difficult to model in mice. We demonstrate premature neuronal differentiation in patient organoids, a defect that could help to explain the disease phenotype.
We then initiate the development of more organoid-based human disease models.41 We were able to reproduce the events leading to human brain cancer formation by electroporating mutagenic DNA constructs and introducing brain cancer specific mutations. Importantly, the new methodology some of the key events in human brain cancer, like the invasive nature of cancer cells, to be replicated in vitro.41 We demonstrated the usefulness of our cancer models for drug treatment by inhibiting tumor growth using EGFR inhibitors and predicting drug effects in a patient and tumor type specific manner. Cerebral organoids become very useful for elucidating and characterizing the teratogenic effects of the ZIKA virus and for predicting its mechanism of infection.42,43 In an attempt to extend those observations, we model the pathology of Herpes Simplex Virus in organoids. We succeed in recreating the pathology and could identify a potential therapeutic strategy for eliminating the virus from the fetal brain.44 We therefore convincingly show that three-dimensional organoids can recapitulate development and disease even in this most complex human tissue.
While we agree that organoids enable disease modeling in complex and structured human tissue, in vitro, like most 3D models, they lack sufficient oxygen supply, leading to cellular stress. We hypothesize that drawbacks might prevent proper lineage commitment. We therefore set out to analyze brain organoid and fetal single cell RNA sequencing (scRNAseq) data using our own and other’s datasets totaling over 190,000 cells. We describe a unique stress signature found in all organoid samples, but not in fetal samples. We demonstrate that cell stress is limited to a defined organoid cell population, and develop Gruffi, an algorithm that uses granular functional filtering to filter out stressed cells from any organoid scRNAseq dataset in an unbiased manner.45 In this way, we offer a robust way to bioinformatically control for the adverse effects of cell stress thereby improving developmental trajectories and strengthening resemblance to fetal data.
In parallel we develop variable organoid protocols that permit us to study different parts of brain development. We push the boundaries by achieving the faithful reproduction of long-range neuron migration in the human brain by assembling dorsally and ventrally patterned organoids (Fig. 6). We recreated a polarity axis and demonstrate that this axis is maintained throughout the organoid culture, leading to correct interactions between the two brain parts. Like in the real human cortex, interneurons within these cultures correctly migrated from the ventral to the dorsal part allowing us to investigate their migration in real time and to test the effect of specific signaling pathways by using specific inhibitors.46
We and others share our appreciation for what seems to be a new technology with enormous potential. We now have a versatile tool that can be coupled to genetic screening permitting us distinct bioassays to unravel novel, disease-associated phenotypes and mechanisms. It comes as no surprise that we are enthusiastically combining decades of multi-disciplinary research outcomes and organoid technology to investigate what other model organisms helped frame the hypotheses on. In recent years, multiple breakthrough discoveries are made, and groundbreaking methodologies are developed. The most prominent of these is the development of the CRISPR-LICHT approach which is a method for genetic screening in 3D organoid systems that can now be applied to any set of human disease genes and any organoid system (Fig. 7).47 The development of the CRISPR−Cas9 endonuclease technology has made diverse methods of genetic engineering readily available to all researchers.48–51 Unlike the previous technologies, the Cas9 endonuclease is guided to the genomic sequence of interest as a means to generate a DSB by a guide RNA sequence (gRNA), making the system highly versatile and easy to apply.52 We combined CRISPR/LICHT, CRISPR/Cas9 dropout screening with lineage barcoding to overcome the intrinsic variability of organoids (Fig. 7). This genetic loss-of-function screening within the modern era of organoid technology allows us to search through sets of genes that are suspected to be involved in a specific human brain disorder.47 We gather definite proof of gene-specific disease relevance while generating an organoid disease model that can be further exploited by the community to portray the disease mechanism or test therapeutic targets. The CRISPR-LICHT technology permits us to establish a mathematical model for organoid growth and to perform a statistical power analysis for the screen to define its scale and interpret its result. Using the methodology, we screened through 173 candidate microcephaly genes, identifying the unfolded protein response as a new process determining brain size in humans. Not only are we able to identify microcephaly genes with CRISPR-LICHT, but we also pinpoint a specific mechanism involved in controlling the size of the brain. The endoplasmic reticulum (ER) was identified as a main hub in controlling extracellular matrix protein secretion (Fig. 8). This mechanism affects the integrity of the tissue, and thus the brain size, and was identified as one cause of microcephaly.
The speed of discoveries is gathering momentum and we seem to be reaching one goal after the other with what appears to be effortless poise but which I can attest to being the merited success of many dedicated and charismatic scientists that lend their talent to stem cell research. It is true that the hypothesis-driven scientist fearlessly tests the status quo putting knowledge to practice and creating platforms for what will be the next ordinary or extraordinary step when claiming the unknown. As we approach the present times there is one more discovery we eagerly share. While using cerebral organoids to show that overproduction of mid-gestational human interneurons causes Tuberous Sclerosis Complex (TSC), a severe neuro-developmental disorder, we identify a previously uncharacterized population of caudal late interneuron progenitors, the CLIP-cells (Fig. 9).53 We show that developmental processes specific to humans are responsible for malformations of cortical development (MCDs), which result in developmental delay and epilepsy in children. In TSC, CLIP cells over-proliferate, generating excessive interneurons, brain tumors, and cortical malformations (Fig. 10). Epidermal growth factor receptor inhibition reduces tumor burden, identifying potential treatment options for TSC and related disorders. The identification of CLIP cells reveals the extended interneuron generation in the human brain as a vulnerability for disease. In addition, this work demonstrates that analyzing MCDs can reveal fundamental insights into human-specific aspects of brain development. We predict that this work will have a long-lasting fundamental impact on the entire field of brain research and will sooner or later find its place in freshly updated textbooks on the disease.
Concluding remarks
The brain, like no other organ, births all thoughts and involuntary triggers. As a consequence it is a treasure chest storing cardinal information; hard to penetrate, retrieve and even understand. It is perhaps not random that many neuroscientists have originally studied philosophy while others moved from bench science to questioning the mind, its powers and limitations, its flexibility to expand and collapse on the path to enlightenment. Such dynamic processes hosted within a tissue that is highly organized and almost fragile.
Human brain development is therefore, correctly described as a complex series of dynamic and adaptive processes that are genetically determined and environmentally influenced and which operate throughout development finally resulting in an organ that is responsible for the widest array of functions we know to exist. Attempting to decipher the genetic codes and molecular pathways that govern cellular function during brain development has been a long and arduous process. The discovery and characterization of stem cells revealed the tangles within the process while instrumentally influencing our understanding by offering the opportunity to make stem cell attributes our asset. Taking advantage of their self-organizing ability, we built a model that was the missing link between knowledge acquired from animal models and the mystery of the human brain.
Born from the passion for evolving science to understand life, methodological developments have always been central to overcoming seemingly impossible obstacles turning improbable outcomes to acquired knowledge. Our lab has significantly contributed in shaping contemporary and future research on brain development in health and disease by careful, almost pedantic experimentalism, and with concentrated focus on multi-disciplinary, expansive and strategic research that pushes the boundaries daring to address the big open questions. At the heels of brain organoid technology, we intertwined quick and efficient gene editing to model diseases that were intractable in mice, including forms of microcephaly and brain cancer. The comparable complexity between human cerebral organoids and primary tissue promises to further our grasp of human-specific complex diseases such as autism and epilepsy but also as patient-specific in vitro cancer models, and for predictive drug testing. Single-cell technologies enhanced our ability to analyze molecular phenotypes at cellular resolution and detect emergent phenotypes that are difficult to tease out with traditional investigation methods.54 Further adaptation of diverse single-cell -omics technologies to brain organoids expanded the set of discernable phenotypes, otherwise hidden, such as novel cell types and/or states that are altered during disease like in our microcephaly and tuberous sclerosis models. Without a doubt the field of brain organoid research is still young, but its applicability, diversity and potential encourage expedient progress. Given the rapid technical advances in the field, we believe that human organoid systems will provide unprecedented opportunities to improve human health.
To conclude, the brain is an organ that is more understood now than ever before but still to such a narrow degree. Despite the jaw dropping advancements across all scientific disciplines of neuroscience, we remain far away from fully understanding it although we appreciate the circuitous complexity of its nature. Attempting to predict what the path will look like in the future would only prove us wrong but what we know for certain is that, as described in this review, neuroscience research resembles a sequel that always has you wishing for more.
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