Madeline Lancaster

University of Cambridge | Cam · MRC Laboratory of Molecular Biology

Our understanding of the genetics of the human cerebral cortex is limited both in terms of the diversity and the anatomical granularity of brain structural phenotypes. Here we conducted a genome-wide association meta-analysis of 13 structural and diffusion magnetic resonance imaging-derived cortical phenotypes, measured globally and at 180 bilatera...

Our understanding of the genetic architecture of the human cerebral cortex is limited both in terms of the diversity of brain structural phenotypes and the anatomical granularity of their associations with genetic variants. Here, we conducted genome-wide association meta-analysis of 13 structural and diffusion magnetic resonance imaging derived cor...

Brain organoids, or cerebral organoids, are a promising new tool to investigate neurodevelopment and disease in a human context. A variety of methods exist with varying degrees of cellular complexity, tissue structure, and homogeneity. Here, we describe self-organizing and self-patterning cerebral organoids. We focus on the most up-to-date and opti...

Self-organizing three-dimensional cellular models derived from human pluripotent stem cells or primary tissue have great potential to provide insights into how the human nervous system develops, what makes it unique and how disorders of the nervous system arise, progress and could be treated. Here, to facilitate progress and improve communication w...

The substantial phenotypic heterogeneity in autism limits our understanding of its genetic etiology. To address this gap, here we investigated genetic differences between autistic individuals ( n max = 12,893) based on core and associated features of autism, co-occurring developmental disabilities and sex. We conducted a comprehensive factor analys...

The biological basis of male–female brain differences has been difficult to elucidate in humans. The most notable morphological difference is size, with male individuals having on average a larger brain than female individuals1,2, but a mechanistic understanding of how this difference arises remains unknown. Here we use brain organoids³ to show tha...

During brain development, axons must extend over great distances in a relatively short amount of time. How the subcellular architecture of the growing axon sustains the requirements for such rapid build-up of cellular constituents has remained elusive. Human axons have been particularly poorly accessible to imaging at high resolution in a near-nati...

Greater understanding of the events preceding neurodegeneration is needed to design effective preventive and therapeutic strategies. In this issue of Cell, Bowles et al. (2021) report cerebral organoids that reveal early events in frontotemporal dementia pathogenesis due to mutations in microtubule-associated protein tau (MAPT), shedding light on a...

The choroid plexus is central to normal brain function by secreting cerebrospinal fluid and dynamically regulating its composition throughout development and homoeostasis. Much of our current understanding of this region of the brain comes from studies in animal models. These fundamental studies have shed light on choroid plexus mechanisms of secre...

Studying the function of common genetic variants in primary human tissues and during development is challenging. To address this, we use an efficient multiplexing strategy to differentiate 215 human induced pluripotent stem cell (iPSC) lines toward a midbrain neural fate, including dopaminergic neurons, and use single-cell RNA sequencing (scRNA-seq...

The human brain has undergone rapid expansion since humans diverged from other great apes, but the mechanism of this human-specific enlargement is still unknown. Here, we use cerebral organoids derived from human, gorilla, and chimpanzee cells to study developmental mechanisms driving evolutionary brain expansion. We find that neuroepithelial diffe...

Cerebral organoids, or brain organoids, can be generated from a wide array of emerging technologies for modeling brain development and disease. The fact that they are cultured in vitro makes them easily accessible both genetically and for live assays such as fluorescence imaging. In this Protocol Extension, we describe a modified version of our ori...

Brain development is an extraordinarily complex process achieved through the spatially and temporally regulated release of key patterning factors. In vitro neurodevelopmental models seek to mimic these processes to recapitulate the steps of tissue fate acquisition and morphogenesis. Classic two-dimensional neural cultures present higher homogeneity...

In recent years considerable progress has been made in the development of faithful procedures for the differentiation of human pluripotent stem cells (hPSCs). An important step in this direction has also been the derivation of organoids. This technology generally relies on traditional three-dimensional culture techniques that exploit cell-autonomou...

The presence of male-female brain differences has long been a controversial topic. Yet simply negating the existence of biological differences has detrimental consequences for all sexes and genders, particularly for the development of accurate diagnostic tools, effective drugs and understanding of disease. The most well-established morphological di...

Coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus, leads to respiratory symptoms that can be fatal. However, neurological symptoms have also been observed in some patients. The cause of these complications is currently unknown. Here, we use human-pluripotent-stem-cell-derived brain...

Coronavirus disease-19 (COVID-19), caused by the SARS-CoV-2 virus, leads primarily to respiratory symptoms that can be fatal, particularly in at risk individuals. However, neurological symptoms have also been observed in patients, including headache, seizures, stroke, and fatigue. The cause of these complications is not yet known, and whether they...

During brain development, human axons must extend over great distances in a relatively short amount of time. How the subcellular architecture of the growing axon sustains the requirements for such rapid build-up of cellular constituents has remained elusive. Human axons have been particularly inaccessible to imaging at molecular resolution in a nea...

The human brain has undergone rapid expansion since humans diverged from other great apes, but the mechanism of this human-specific enlargement is still unknown. Here, we use cerebral organoids derived from human, gorilla and chimpanzee cells to study developmental mechanisms driving evolutionary brain expansion. We find that the differentiation of...

Brain barrier and support in a dish Deep within the brain, the choroid plexus filters blood and secretes cerebrospinal fluid (CSF), a nutrient-rich liquid that bathes and supports the brain and protects it from entry of toxic compounds. Current understanding of this vital tissue in humans is limited. Pellegrini et al. developed choroid plexus organ...

Common genetic variants can have profound effects on cellular function, but studying these effects in primary human tissue samples and during development is challenging. Human induced pluripotent stem cell (iPSC) technology holds great promise for assessing these effects across different differentiation contexts. Here, we use an efficient pooling s...

The human brain is often described as the most complex organ in our body. Because of the limited accessibility of living brain tissue, human-specific features of neurodevelopment and disease remain largely unknown. The ability of induced pluripotent stem cells to self-organize into 3D brain organoids has revolutionized approaches to studying brain...

The past decade has seen an explosion in the field of in vitro disease modelling, in particular the development of organoids. These self-organizing tissues derived from stem cells provide a unique system to examine mechanisms ranging from organ development to homeostasis and disease. Because organoids develop according to intrinsic developmental pr...

Neural organoids have the potential to improve our understanding of human brain development and neurological disorders. However, it remains to be seen whether these tissues can model circuit formation with functional neuronal output. Here we have adapted air–liquid interface culture to cerebral organoids, leading to improved neuronal survival and a...

Membrane potential is well-studied in neuronal physiology, but its role in brain development and cell fate determination is less well understood. In two recent studies, bioelectric properties of progenitors and migrating neurons are shown to be tightly regulated, and their disruption leads to abnormalities in fate determination and neuronal positio...

The original version of this paper contained an incorrect primer sequence. In the Methods subsection “Rampage libraries,” the text for modification 3 stated that the reverse primer used for library indexing was 5′-CAAGCAGAAGACGGCATACGAGATXXXXXXXXGTGACTGGAGT-3′. The correct sequence of the oligonucleotide used is 5′-CAAGCAGAAGACGGCATACGAGATXXXXXXXXG...

The field of developmental neuroscience is benefitting from recent technological advances that allow access to organogenesis in vitro via organoid preparations. These methods have been applied to better understanding neural identity, and have opened up a window into the early events that occur during development of the human brain. However, current...

This corrects the article DOI: 10.1038/nbt.3906.

Specialized RNA-seq methods are required to identify the 5' ends of transcripts, which are critical for studies of gene regulation, but these methods have not been systematically benchmarked. We directly compared six such methods, including the performance of five methods on a single human cellular RNA sample and a new spike-in RNA assay that helps...

Three-dimensional neural organoids are emerging tools with the potential for improving our understanding of human brain development and neurological disorders. Recent advances in this field have demonstrated their capacity to model neurogenesis, neuronal migration and positioning, and even response to sensory input. However, it remains to be seen w...

In large mammalian brains, including those of humans, the surface of the cortex is highly folded. How these convolutions form is still unclear, but recent work in Nature Physics by Karzbrun et al. (2018) supports a mechanism involving differential surface swelling combined with internal constraint. In large mammalian brains, including those of huma...

Transplantation of cerebral organoids into the mouse brain overcomes some limitations of in vitro systems.

Steroids have an important role in growth, development, sexual differentiation and reproduction. All four classes of steroids, androgens, estrogens, progestogens and glucocorticoids, have varying effects on the brain. Androgens and estrogens are involved in the sexual differentiation of the brain, and also influence cognition. Progestogens such as...

Model organisms are widely used in research as accessible and convenient systems to study a particular area or question in biology. Traditionally only a handful of organisms have been widely studied, but modern research tools are enabling researchers to extend the set of model organisms to include less-studied and more unusual systems. This Forum h...

Three-dimensional cell culture models have either relied on the self-organizing properties of mammalian cells or used bioengineered constructs to arrange cells in an organ-like configuration. While self-organizing organoids excel at recapitulating early developmental events, bioengineered constructs reproducibly generate desired tissue architecture...

3D brain organoids are a powerful tool with prospective application for the study of neural development and disease. Here we describe the growth factor-free method of generating cerebral organoids from feeder-dependent or feeder-free human pluripotent stem cells using standard laboratory equipment. The protocol outlined below allows generation of 3...

Cerebral organoids recapitulate human brain development at a considerable level of detail, even in the absence of externally added signaling factors. The patterning events driving this self-organization are currently unknown. Here, we examine the developmental and differentiative capacity of cerebral organoids. Focusing on forebrain regions, we dem...

Expansion of the neocortex is thought to underpin the higher cognitive abilities of a number of mammalian lineages, such as cetaceans, elephants, and primates, with humans exhibiting a particularly enlarged and dense cerebral cortex. However, the evolutionary and developmental mechanisms that led to this expansion are not well-understood and limite...

Madeline Lancaster received her first degree in biochemistry from the Occidental College in Los Angeles, California, and continued to follow her interests by pursuing a PhD in biomedical sciences in the laboratory of Joseph Gleeson at University of California, San Diego. She then left the USA to train as a postdoctoral fellow with Jürgen Knoblich a...

Organoids derived from human pluripotent stem cells recapitulate the early three-dimensional organization of the human brain, but whether they establish the epigenomic and transcriptional programs essential for brain development is unknown. We compared epigenomic and regulatory features in cerebral organoids and human fetal brain, using genome-wide...

An expansion of the cerebral neocortex is thought to be the foundation for the unique intellectual abilities of humans. It has been suggested that an increase in the proliferative potential of neural progenitors (NPs) underlies the expansion of the cortex and its convoluted appearance. Here we show that increasing NP proliferation induces expansion...

The ability to model human brain development in vitro represents an important step in our study of developmental processes and neurological disorders. Protocols that utilize human embryonic and induced pluripotent stem cells can now generate organoids which faithfully recapitulate, on a cell-biological and gene expression level, the early period of...

Recent breakthroughs in pluripotent stem cell technologies have enabled a new class of in vitro systems for functional modeling of human brain development. These advances, in combination with improvements in neural differentiation methods, allow the generation of in vitro systems that reproduce many in vivo features of the brain with remarkable sim...

Recently emerging methodology for generating human tissues in vitro has the potential to revolutionize drug discovery and disease research. Currently, three-dimensional cell culture models either rely on the pronounced ability of mammalian cells to self organize in vitro ¹⁻⁶ , or use bioengineered constructs to arrange cells in an organ-like config...

Significance We have used single-cell RNA sequencing to compare human cerebral organoids and fetal neocortex. We find that, with relatively few exceptions, cells in organoid cortex-like regions use genetic programs very similar to fetal tissue to generate a structured cerebral cortex. Our study is of interest, as it shows which genetic features und...

As a group, we met to discuss the current challenges for creating meaningful patient-specific in vitro models to study brain disorders. Although the convergence of findings between laboratories and patient cohorts provided us confidence and optimism that hiPSC-based platforms will inform future drug discovery efforts, a number of critical technical...

Human brain development exhibits several unique aspects, such as increased complexity and expansion of neuronal output, that have proven difficult to study in model organisms. As a result, in vitro approaches to model human brain development and disease are an intense area of research. Here we describe a recently established protocol for generating...

Classical experiments performed half a century ago demonstrated the immense self-organizing capacity of vertebrate cells. Even after complete dissociation, cells can reaggregate and reconstruct the original architecture of an organ. More recently, this outstanding feature was used to rebuild organ parts or even complete organs from tissue or embryo...

The complexity of the human brain has made it difficult to study many brain disorders in model organisms, highlighting the need for an in vitro model of human brain development. Here we have developed a human pluripotent stem cell-derived three-dimensional organoid culture system, termed cerebral organoids, that develop various discrete, although i...

Captions for Supplementary Videos

Supplementary Video 2

Supplementary Video 1

Extended Data Figure 1. Generation of cerebral organoids from multiple human pluripotent stem cells. a. Hemotoxylin-eosin staining of cerebral organoids compared with stationary culture reveals overall larger tissues with substructure reminiscent of brain regions such as forebrain cortex (arrows) and choroid plexus (arrowhead). b. Higher magnificat...

Supplementary Video 3

Extended Data Figure 2. Neural identity during differentiation of cerebral organoids. a. RT-PCR for the pluripotency markers Oct4 and Nanog as well as neural identity markers Sox1 and Pax6 in undifferentiated human ES cells and following differentiation at 9 days revealing induction of neural identity with decreased pluripotent identity at 9 days o...

Extended Data Figure 3. Radial glial organization and morphology. a. Staining for the chromatin remodeling BAF components Baf53a (green, upper panels) and Baf53b (green, lower panels) in serial sections of the same tissue showing the neural progenitor-specific Baf53a expressed in VZ RGs while the neuron-specific Baf53b is expressed in DCX+ (red) ne...

Extended Data Figure 5. Human features of cortical development not recapitulated in mouse organoids. a. Low magnification image of the region shown in Figure 5a revealing the presence of a separated region of oRGs (demarcated by arrowheads) that appear separate from the VZ in all regions (brackets) but more separated and with a layer of Tuj1+ fiber...

Extended Data Figure 4. Spatial organization and characteristics of cortical neuron identities. a. Staining for the preplate marker Tbr1 (green) and the deep-layer marker Ctip2 (red) at day 30 revealing rudimentary spatial separation reminiscent of the early stages of CP development. b. Immunohistochemistry for the early born neuron marker Ctip2 (g...

Extended Data Figure 8. shRNA mediated knockdown of CDK5RAP2 in human organoids. a. Western blot for endogenous CDK5RAP2 in 293T cells transfected with 4 different shRNAs against CDK5RAP2. shRNA1 and 2 are most efficient while shRNA 4 leads to a modest reduction in protein. Alpha-Tubulin is shown as a loading control. b. Higher magnification of res...

Extended Data Figure 6. Patient growth parameters. a. All growth parameters were significantly reduced both at birth and postnatally, with all z-scores less than −2 standard deviations from the population mean for age and sex (dashed line). Weight (wgt), height (hgt) and head circumference (occipitofrontal circumference, ofc) at birth and at curren...

Extended Data Figure 7. Characterization of patient derived iPSCs and cerebral organoids. a. iPS cells derived from A3842 patient skin fibroblasts exhibit typical ES cell-like morphology. Four lines were chosen for analysis based on this typical morphology and pluripotency. b. Alkaline phosphatase staining (blue) of patient derived iPS cell colonie...

In any mitotic cell, the orientation of the mitotic spindle determines the orientation of the cleavage plane and therefore the position of the two daughter cells. When combined with polarization of cellular components, spindle orientation is also a well-conserved means of generating daughter cells with asymmetric cell fates, such as progenitors and...

Mechanisms of signal transduction regulation remain a fundamental question in a variety of biological processes and diseases. Previous evidence indicates that the primary cilium can act as a signalling hub, but its exact role in many of the described pathways has remained elusive. Here, we investigate the mechanism of cilia-mediated regulation of t...

The ciliopathy Joubert syndrome is marked by cerebellar vermis hypoplasia, a phenotype for which the pathogenic mechanism is unclear. To investigate Joubert syndrome pathogenesis, we have examined mice with mutated Ahi1, the first identified Joubert syndrome-associated gene. These mice show cerebellar hypoplasia with a vermis-midline fusion defect...

Supplementary Figure 1. Cilia quantification in MEFs a. Staining for cilia (acetylated tubulin, red, arrow) in serum starved MEFs with reapplication of serum. These results are quantified at the right for at least 200 cells. ***P<0.0005, Chi-square test. Nuclei are labeled with Hoechst (blue). b. Staining for the primary cilium (acetylated tubulin,...

Supplementary Figure 3. β-catenin and Jbn are sequestered in the presence of cilia a. Staining for endogenous β-catenin (green) in ciliated (actylated tubulin, red) and nonciliated (arrow) IMCDs revealing increased nuclear (Hoechst, blue, outlined) localization of β-catenin in nonciliated cells compared with ciliated neighboring cells. b. Quantific...

Supplementary Figure 2. Kif3a knockdown and quantification of ciliation a. Western blot analysis of whole cell lysates from 293Ts transfected with Kif3A siRNA. Alpha-tubulin is shown as a loading control. b. Staining for cilia (acetylated tubulin, red) in MEFs transfected with Kif3a siRNA #3. Arrows point to cilia in control transfected cells. Hoec...

Supplementary Figure 7. Full western blot scans

Supplementary Figure 4. Loss of cilia potentiates Jbn’s role in the Wnt pathway a. 293T cells stained for cilia with antibody to acetylated tubulin (red) display clear cilia with Jbn-GFP (green) localized to the basal body similar to previously described in other ciliated cells2, 38. Hoechst was used to stain the nucleus (blue). b. Cilia staining o...

Supplementary Figure 5. Dnchc2 siRNA and aphidicolin treatment effect on ciliation a. Luciferase activity in MEFs without serum starvation and transfected with 5 pmol Dnchc2 siRNA and treated with WCM or LCM. N.S.=not significant, n=3 independent experiments, Student’s t-test. Error bars represent S.E.M. b. Staining for cilia (acetylated tubulin, r...

Supplementary Figure 6. Dnchc2 loss leads to varying effects on cilia and Wnt signaling a. Whole mount images of X-gal stained E9.5 control and Dnchc2 mutant embryos revealing increased Wnt activity in the mid-hindbrain region and nasal folds (arrows). b. β-galactosidase staining (green) of E9.5 BATgal+ and BATgal− control mesonephros (outlined). H...

Wnt signaling encompasses a variety of signaling cascades that can be activated by secreted Wnt ligands. Two such pathways, the canonical or beta-catenin pathway and the planar cell polarity (PCP) pathway, have recently received attention for their roles in multiple cellular processes within the kidney. Both of these pathways are important for kidn...

Degeneration of photoreceptors is a common feature of ciliopathies, owing to the importance of the specialized ciliary structure of these cells. Mutations in AHI1, which encodes a cilium-localized protein, have been shown to cause a form of Joubert syndrome that is highly penetrant for retinal degeneration. We show that Ahi1-null mice fail to form...