Neural Stem Cells

Organoids and Human Translation

May 4-11, 2024

Director: Lorenz Studer

Memorial Sloan Kettering Cancer Center, New York, USA

Faculty:

Jürgen Knoblich, IMBA, Vienna, Austria & Institute of Human Biology, Base, Switzerland 

Arnold Kriegstein, University of California, San Francisco, USA 

Marius Wernig, Stanford University, USA

Fiona Doetsch, University of Basel, Switzerland

Tracy Young-Pearse, Brigham and Women’s Hospital & Harvard Medical School, Boston, USA

Jerome Mertens, University of California, San Diego, USA

Lorenz Studer, Memorial Sloan Kettering Cancer Center, New York, USA

Over the last few years, there has been enormous progress in using stem cells to study the development and function of the nervous system and to pursue applications in modelling and treating neural diseases. Particularly notable are advances in single-cell genomics that have revolutionized our understanding of cell heterogeneity and gene regulatory networks within the developing CNS and have revealed species-specific differences in cell composition and identity. Similarly, heterogeneity within the adult neural stem cell (NSC) compartment has uncovered novel NSC functional states in response to various physiological conditions ranging from exercise to pregnancy, regeneration, ageing and brain repair. Another major development has been the rapid progress in neural organoid technologies that have enabled functional studies directly in human cells. Furthermore, human pluripotent stem cell (hPSC)-based organoids can be combined into complex assembloids to build CNS circuitry in vitro or grafted into the adult CNS to study functional integration and neuroimmune interactions in vivo. Despite such progress, many important technical challenges remain, such as the need for further improvements in robustness and scalability of organoid-based culture systems and for establishing conditions appropriate to capture adult or aged-like properties of the human CNS.

Another exciting development is the availability of well-annotated patient cohorts and longitudinal datasets for both neurodevelopmental and neurodegenerative disorders, which enable new classes of patient-specific disease models. Conditions have been established to capture neural disease-related phenotypes across large numbers of hPSC lines and link those to patient stratification, disease progression, or therapeutic responses. Finally,  we are at an exciting time point in the development of cell-based therapies to the CNS, where after decades of gradual progress, several hPSC-based cell products have reached clinical testing. Those include dopamine neuron replacement for Parkinson’s disease or the treatment of intractable seizures using hPSC-derived interneurons, with several additional cell types and promising disease targets under development.

This Advanced Course will cover many of those major developments as presented by leaders in the field, and it will offer a unique perspective on the future of stem cell-based approaches in studying brain function and disease.

Arnold Kriegstein

Genomic Insights into Early Human Brain Development Relevant to Organoid Models

The human cerebral cortex is more than three times expanded compared to our closest non-human primate relatives. The cortex emerges from an initially pseudostratified neuroepithelium that gives rise to radial glia, the neural stem cells of the cortex. A number of subtypes of radial glia have been identified in human cortical development, and single cell RNA sequencing (scRNAseq) has contributed to a novel model of primate corticogenesis, highlighted human-specific features of cortical development and providing a benchmark for in vitro organoid models of brain development and disease. We have begun to characterize the molecular populations of cellular subtypes that exist during human cortical development in prenatal and postnatal stages. We find that major signaling pathways including Notch, Wnt, and mTOR drive the specification and maintenance of neuroepithelial stem cells and radial glia. We also identify critical periods of differentiation and maturation for the major classes of excitatory and inhibitory neurons, as well as glial cell types and pinpoint gender- and region-specific developmental programs. We identify cell types and developmental stages most vulnerable to genetic insults in neurodevelopmental disorders and demonstrate sex-specific transcriptomic events that can explain skewed male to female risk ratios for neurodevelopmental diseases. We have also profiled synaptic proteins during cortical development in human, macaque and mouse and identified human specific mechanisms leading to neoteny of cortical synapse maturation in the perinatal period. In addition, we utilize brain organoids to model cortical development and neurodevelopmental disease and have recently examined syndromic forms of autism using organoid models as well as postmortem human brain samples to explore convergent pathways.

Recent studies have begun to utilize brain organoids to model cortical development and neurodevelopmental disease, however the extent to which developmental processes are accurately represented in organoids is currently unclear, and reported limitations regarding structural organization, cellular health, and the stability of the cultures over time have demonstrated the need for a thorough comparison between organoids and primary cells. We have performed a direct comparison of cortical organoids and primary human cortical samples throughout neurogenesis using single cell RNA sequencing and complementary immunohistochemical analyses. We find that the specificity of cellular subtype is not as clearly resolved in organoids as during normal development. This lack of specificity in crisp cellular identity may be problematic for maturation of these cells. Interestingly, differentially expressed genes enriched in organoids highlight pathways reflective of metabolic and ER stress. Although organoids are a powerful model system, a better definition of their limitations and how best to utilize these models is required as we strive to both improve our understanding of the brain and how best to study neurodevelopmental diseases.

Fiona Doetsch

Stem Cells in the Adult Brain: Identity, Regulation and Heterogeneity

Neural stem cells reside in specialized niches in the adult mammalian brain. Adult neural stem cells dynamically integrate intrinsic and extrinsic signals to either maintain the quiescent state or to become activated to divide and generate neurons and glia. Recent findings have uncovered unexpected heterogeneity amongst adult neural stem cells, which are shedding new light on our understanding of adult neural stem cells. We will explore the history of adult neurogenesis as well as current perspectives and novel insights gained using recent technological advances. Topics to be covered include the identity of adult neural stem cells, their developmental origins, in vivo lineages and cell states, and regulation in different physiological states. We will also explore how stem cells are regulated by different compartments in the niche, including local cell-cell interactions and long-range signals, such as neural circuits, the vasculature and systemic factors, as well as signals in the cerebrospinal fluid. Finally, we will discuss important new advances in understanding the functional relevance of adult neural stem cell heterogeneity for on demand-neurogenesis and gliogenesis for adaptive brain plasticity.

Marius Wernig

How to make a neuron and treat the brain

Cellular differentiation and lineage commitment are considered robust and irreversible processes during development. Challenging this view, we found that expression of only three neural lineage-specific transcription factors Ascl1, Myt1l, and Brn2 could directly convert mouse fibroblasts into functional in vitro. These induced neuronal (iN) cells expressed multiple neuron-specific proteins, generated action potentials, and formed functional synapses. Thus, iN cells are bona fide functional neurons.

Unlike reprogramming towards other lineages, such as iPS cell reprogramming, the iN cell reprogramming process is very efficient (up to 20%) and deterministic. Exploring the underlying mechanisms, we identified a molecular explanation for the high reprogramming efficiency: We discovered that Ascl1, a transcriptional activator, acts as an “on target” pioneer factor, i.e. it has a unique property to access its physiological targets in fibroblasts even though these sites are in a closed chromatin state, and considered inaccessible for transcription factors. In this way, Ascl1 can directly and robustly induce the neuronal transcriptional program. Measuring the chromatin state using transposase accessibility called ATAC-Seq, we further found that Ascl1 rapidly remodels the chromatin at its target sites into a stable nucleosome-free region, which is surrounded by two flanking nucleosomes. Surprisingly, Ascl1 alone is sufficient to induce fully functional iN cells, but in the majority of cells activates also non-neuronal programs.

An important question in cell fate specification is how a cell identity can be maintained after it has been established. Intriguingly, Ascl1 and equivalent proneural bHLH transcription factors are only transiently expressed and turned off as neurons mature into postmitotic cells. Thus, the maintenance is likely regulated by independent mechanisms. We observed that Myt1l, a zinc finger domain protein mutated in autism-related syndromes, primarily functions as a transcriptional repressor, suppressing the fibroblast and other non-neuronal programs during iN cell reprogramming. This suggests that the physiological role of Myt1l is to ensure the maintenance of neuronal identity by repressing many transcriptional programs except neuronal genes, thereby functioning in exactly the inverse way as REST, which blocks neuronal genes in many non-neuronal cell types.

Finally, we seek to explore new avenues for cell therapies in the brain. Microglia, as the central nervous system’s main immune cell type, are considered major players in contributing to the pathogenesis of many neurological diseases. We have investigated the use of hematopoietic cell transplantation using primary bone marrow cells or human induced pluripotent stem (iPS) cell-derived cells and developed methods to replace endogenous microglia with bone marrow- or iPS cell-derived cells. We found proof-of-concept evidence that such microglia replacement can have therapeutic effects in models of neurodegeneration and neuroinflammation. In conclusion, combining cellular reprogramming and gene editing is a powerful approach to developing next-generation cell therapies for the brain.

Tracy Young-Pearse

Part I: Modeling genetic risk and resilience to neurological disease using induced pluripotent stem cells 
Part II: Interrogation of person-specific molecular mechanisms underlying Alzheimer’s disease using stem cell technology.

Clinicians and pathologists have long recognized that late-onset Alzheimer’s disease (LOAD) manifests along a spectrum of cognitive deficits and levels of neuropathology. Although AD is somewhat stereotyped in terms of both the symptoms of cognitive dysfunction with age and the patterns of pathological aggregation of amyloid β (Aβ) and tau in the brain, Alzheimer’s dementia is heterogeneous in many other ways. There exists a range of ages of onset and rates of progression, differences in the cognitive domains that are first affected, and a wide range in both the abundance and variety of neuropathological lesions. Until recently, studies of AD in model systems, including human-derived cell culture models and rodent models expressing pathogenic transgenes, relied on comparisons between ‘AD’ and ‘not AD’. However, this binary segregation ignores the heterogeneity in our aging population with regards to the accumulation of amyloid plaques, tau tangles, and other protein aggregates and the slope of cognitive abilities with age. As a result, our ability to develop effective therapies is greatly impaired by the loss of power to disentangle different molecular roads that lead to AD. The starting points for these different molecular roads are, in part, rooted in combinations of genetic risk and resilience factors that vary in different individuals. Genetic studies support the idea of heterogeneous disease mechanisms, with around 70 associated loci to date that implicate several biological processes that mediate risk for AD. In turn, combinations of different genetic risk variants may be converging on different interrelated biological domains that ultimately converge on synapse loss and degeneration. Human-induced pluripotent stem cell (iPSC) technology offers a versatile platform that not only captures genetic susceptibility and resilience factors but also serves as a controlled and manipulable experimental system for assessing biological domain dysfunction and response to intervention.

Longitudinal studies in human populations undergoing deep phenotypic characterization are particularly informative in defining the heterogeneity in neuropathological and cognitive outcomes with age. One of the longest-running and most deeply studied cohorts of ageing are the Religious Orders Study (ROS) and Memory and Aging Project (MAP) of Rush University, which have enrolled over 3,000 individuals at the age of 65 years or above. The participants are followed longitudinally with annual quantitative cognitive testing. Following death, their brain tissue is analyzed by neuropathologists to acquire quantitative measurements of amyloid plaques, tau tangles, Lewy bodies, TDP43-positive inclusions, and vascular lesions. Across the population, there is a striking variety of mixed cognition-related neuropathological phenotypes present and a wide range of cognitive outcomes. In addition to neuropathological analyses, omics profiles are also obtained from ROSMAP brain tissue, including whole genome sequencing, bulk and single nucleus RNA-sequencing (RNA-seq), proteomic profiling by tandem mass tag-mass spectrometry (TMT-MS), and lipidomic profiling. Analyses of these data also show disruption of different biological domains across individuals with AD. These data argue strongly for the utility of experimental systems that can capture the molecular underpinnings of this phenotypic diversity.

We recently developed iPSC lines from over 100 individuals from the ROS and MAP cohorts that span the cognitive and neuropathological spectrum of ageing. There are a variety of experimental approaches for utilizing large sets of deeply phenotyped cohorts of iPSC lines such as these for interrogating mechanisms of neurological diseases downstream of genetic risk and resilience factors. In this Advanced Course, a range of different approaches will be presented, with specific examples provided from our efforts focused on disentangling the molecular roads that lead to Alzheimer’s disease. We will describe how each of these experimental systems – monocultures of single cell types, multicellular co-cultures of different cell types, organoids, and brain-on-chip systems – have their utility in studying disease pathogenesis. New findings from the AD field will be presented that arose from efforts combining genome engineering approaches with studies of natural genetic variation using iPSC technology.