Organoids and Neural Reprogramming for Disease Modeling
May 3-10, 2025
Director: Marius Wernig
Stanford University, USA
Faculty:
Kristin Baldwin, Columbia University, New York, USA
Elena Cattaneo, University of Milan, Italy
Anders Björklund, Lund University, Sweden
Jürgen Knoblich, Institute of Molecular Pathology, Vienna Biocenter, Austria
Michael Rape, University of California, Berkeley, USA
Elly Tanaka, Institute of Molecular Pathology, Vienna Biocenter, Austria
Marius Wernig, Stanford University, USA
Neural reprogramming is a groundbreaking advancement for neuroscience that allows scientists to efficiently generate specialized neural cells from patient’s skin cells that closely mimic the structure and function of the main cellular components of the central nervous system (CNS). CNS organoids derived from induced pluripotent stem cells (iPSCs) represent another key innovation to provide scientists the opportunity to study three-dimensional developmental processes in young organoids and observe the maturation of reprogrammed CNS cell types in three dimensions in old organoids. By combining CNS building blocks in defined ways, such models interrogate not only the cell biology on the single-cell type level but enable the dissection of more complex cell-cell interactions that are critical to understanding brain homeostasis and ongoing disease processes. The investigation of intricate interactions between CNS cell types remained a challenge that the field is now on the cusp of addressing.
The Advanced Course will be taught by renowned scientists in their fields. The lectures will cover basic developmental concepts underlying neural reprogramming and developing CNS organoids, their use to investigate molecular and cellular mechanisms of normal physiology and pathophysiology of neuropsychiatric and neurodegenerative disease, identifying potential new therapeutic strategies, and paving the way for future significant advancements in personalized medicine.
Elena Cattaneo
Exploiting organoids to study genetic brain diseases: Insights from Huntington’s disease
Organoids are powerful tools for modeling neurodevelopmental processes and the mechanisms underlying human brain diseases. They enable the generation of multiple specialized cell types within a single structure, effectively replicating aspects of brain cytoarchitecture. Further advancements in the model have led to the development of brain neuroloids, chimeroids, assembloids, and mosaic organoids. In Huntington’s disease (HD), mosaic organoids have been created by combining cells with different genotypes, allowing for the investigation of both cell-autonomous and non-cell-autonomous components. Maintained in vitro for extended periods, organoids also provide an ideal platform to study the long-term impact of disease modifiers derived from Genome-Wide Association Studies (GWAS) data. We will illustrate this with examples of cis- and trans-modifiers of age of onset identified in HD patients that are revolutionizing the field. Overall, stem cell technologies and 3D models facilitate the interrogation of GWAS data and the evaluation of the effects of single variants on a broad range of neuronal phenotypes. These approaches open new avenues for understanding and potentially treating complex triplet repeat diseases affecting the brain.
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, it also activates 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 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 main immune cell type in the central nervous system, are considered major players in contributing to pathogenesis in 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.
Anders Björklund
Cell-based therapy of neurodegenerative conditions: From neuronal replacement to circuitry repair
Organoids and cellular reprogramming hold promise to open new avenues for treatment of degenerative brain diseases. In my lectures, I will focus on the use of stem cell-derived or reprogrammed cells as tools for repair of neural circuits in the brain. I will discuss how the field of cell-based brain repair has evolved, from the classic studies in the 1970s and 80s using cells obtained from the developing fetal brain to the current approaches using cells derived from embryonic or induced pluripotent stem cells, and discuss the experience gained from the use of dopamine neurons and dopamine neuron progenitors to restore dopamine function in patients with Parkinson´s disease (PD). The clinical trials using dopamine neuron transplants in PD patients have led the field and stimulated efforts to develop cell-based restorative approaches in other neurodegenerative conditions such as Huntington´s disease, injury, stroke and epilepsy. I will review the progress made in these fields and discuss the prospects for the development of cell-based strategies for more refined neural circuitry repair.
Kristin Baldwin
Using Stem Cells to Explore the Genetics Underlying Brain Disease.
Each person’s distinct genetics and environment predispose them to some phenotypes and confer resilience to others. How do all the individual variants across the genetic landscape combine to yield larger phenotypic impacts in aggregate? How does genetic variation govern the penetrance of deleterious mutations, variable expressivity, and pleiotropy? What is the role of the environment across the lifespan? Understanding how these elements interact will advance our knowledge of human development, aging, health, and disease. Our functional genomics approach integrates human-induced pluripotent stem cell models with CRISPR-based genome engineering to introduce and reverse genetic variation, yielding precision models that can be combined with genetic and pharmacological screens. With this approach, we demonstrated that diverse risk variants share downstream convergent impacts and that when added together, their combinatorial perturbations yield novel non-additive outcomes that cannot yet be predicted by individual manipulations alone. We seek to understand the genetic regulation of phenotype and how developmental, cellular, and environmental contexts impact it. Thus, rather than just characterize the impact of trait-associated variants, we seek to uncover modifiers that alter it. For example, we study how genotype-phenotype relationships vary across people and dynamic conditions. Our goal is to decipher the frameworks that buffer genetic risk in order to confer biological resilience and promote healthy development. We are uniquely positioned to answer critical questions: How does the environment impact genetic regulation? Why are there marked sex effects across many human traits and diseases? What are the molecular mechanisms of resilience whereby individuals with high genetic risk show no clinical manifestation of disease? Understanding the basic biology governing the complex interplay between genetic variants and the environment will springboard the development of novel, personalized approaches to improve health and prevent disease.
Elly Tanaka
Self-organization of neural tissue
Neural development and regeneration involve the interaction between cell types, resulting in spatial patterning and diversification of neural progenitors and their descendent neurons. These patterning systems are robust to variation and cell removal. From the stem cell/organoid point of view, patterned neural organoids can self-organize from pluripotent stem cells. In our lab, we have shown that neural tube organoids self-organize in response to a pulse of retinoic acid, which causes induction of FoxA2 and acceleration of neural commitment. These lectures will focus on the cellular and molecular mechanisms of spinal cord self-organization of stem cells in pluripotent stem cell-derived organoids and in regenerating spinal cord tissue in axolotl.
Michael Rapé
From basic discoveries to new therapeutic modalities: Stress signalling in neurodegeneration
Neuronal development and homeostasis can withstand mutational or environmental insults. Key to resilient neuronal cell fate specification are signalling pathways that detect stresses, such as mutations, energy depletion, oxidative damage, or toxin exposure, and, in turn, instigate reactions that alleviate or bypass such conditions. How stress responses safeguard neuronal differentiation and homeostasis is poorly understood. We have recently discovered several stress responses with important roles in neurons, including dimerization quality control or the reductive stress response. These pathways ensure neuronal homeostasis by controlling processes as diverse as complex formation, protein aggregation, and energy generation. However, while transient stress signalling provides cells with time to repair the damage, these pathways must also be turned off at the right time and place to prevent tissue degeneration. How stress response pathways are terminated is not known.
Our discovery of a stress response pathway required for efficient neuronal reprogramming will be a primary focus of discussion. In addition, attention will be devoted to regulated stress response silencing that is required for neuronal survival and, as highlighted by reprogramming experiments, is tightly connected to neurodegenerative disease. Our work suggests novel therapeutic approaches based on targeted protein degradation strategies we spearheaded, which allow us to correct aberrant stress signalling for therapeutic benefit against neurodevelopmental or neurodegenerative disorders.