Modeling Alzheimer's disease using cerebral organoids: Current challenges and prospects

How well do brain organoids capture your brain?

Three-dimensional (3D) stem cell differentiation cultures recently emerged as a novel model system for investigating human embryonic development and disease progression in vitro, complementing existing animal and two-dimensional (2D) cell culture models. Organoids, the 3D self-organizing structures derived from pluripotent or somatic stem cells, can recapitulate many aspects of structural organization and functionality of their in vivo organ counterparts, thus holding great promise for biomedical research and translational applications. Importantly, faithful recapitulation of disease and development processes relies on the ability to modify the genomic contents in organoid cells. The revolutionary genome engineering technologies, CRISPR/Cas9 in particular, enable investigators to generate various reporter cell lines for prompt validation of specific cell lineages as well as to introduce disease-associated mutations for disease modeling. In this review, we provide historical overviews, and discuss technical considerations, and potential future applications of genome engineering in 3D organoid models.

Microcephaly-associated mutations disrupt microtubule-dependent WDR62 translocation from the Golgi complex to the mitotic spindle poles, impair mitotic progression, and alter neurogenic trajectories in patient induced Pluripotent Stem Cell-derived 2D and 3D models of human neurodevelopment.

A wealth of evidence attests that the organs of developing embryos, particularly the developing brain, are acutely sensitive to chemical perturbations. However, scientists know very little about how exposures to specific endogenous chemicals actually impact human development or children’s ability to learn. And there are almost no data on how the vast majority of the 84,000 chemicals currently listed in the Toxic Substances Control Act (TSCA) Inventory1—including most of the 201 compounds known to be neurotoxic to adults and the 1,000 chemicals shown to be neurotoxic to animals2—may affect developing infants. It is also unclear whether testing with animals always provides accurate insights into human developmental susceptibility. A new line of research based on human stem cells is providing important insights into how chemicals may affect neonatal development. Stem cells are the master cells capable of producing some or all of the 200-plus different types of cells in the human body. In time, some researchers believe stem cells may enable scientists to amass far more data on how exposure to environmental chemicals affects human development, particularly the development of the brain. Now is a “critical time to be talking about stem cell research in the environmental health context,” says Tracey Woodruff, director of the Program on Reproductive Health and the Environment at the University of California, San Francisco (UCSF) Medical School.

Adult stem cells retain some capacity for self-renewal, although it is limited in comparison with embryonic stem cells, and are more restricted in their differentiation capacity. Adult stem cells are also commonly referred to as tissue specific stem cells. Some adult stem cells are still able to give rise to several specialized cell types (multipotent stem cells), whereas others are limited to a single specialized cell type (unipotent stem cells). Progenitor cells, the progeny of adult stem cells, differ from stem cells in that the potential for long-term self-renewal is lost. Scientifically, progenitor cells are more differentiated than stem cells. Primary stem cell progeny progenitor cells, usually known as multipotent adult progenitor cells, have full lineage-specific potential, whereas next-generation progenitors (oligopotent progenitors) are more lineage-restricted. This adult stem cell and progenitor cell hierarchy system exists to preserve a homeostatic repair and maintenance of the body, replenishing specialized cells and sustaining the routine cellular turnover in regenerative organs. Furthermore, the properties of these cells make them good candidates for targeted drug/gene delivery to specific organs; for example, mesenchymal stem cells (MSCs) have recently been shown to preferentially home to the lung. Adult stem and progenitor cells may be either circulating or resident in a particular tissue/organ system, including the lung and pulmonary vasculature. This chapter briefly describes the adult stem and progenitor cells currently identified in the pulmonary vasculature and introduces methodological approaches to successfully identify stem and progenitor cells.

Three-dimensional (3D) cerebral cortical organoids are popular in vitro cellular model systems widely used to study human brain development and disease, compared to traditional stem cell-derived methods that use two-dimensional (2D) monolayer cultures. Despite the advancements made in protocol development for cerebral cortical organoid derivation over the past decade, limitations due to biological, mechanistic, and technical variables remain in generating these complex 3D cellular systems. Building from our previously established differentiation system, we have made modifications to our existing 3D cerebral cortical organoid protocol that resolve several of these technical and biological challenges when working with diverse groups of human induced pluripotent stem cell (hiPSC) lines. This improved protocol blends a 2D monolayer culture format for the specification of neural stem cells and expansion of neuroepithelial progenitor cells with a 3D system for improved self-aggregation and subsequent organoid development. Furthermore, this "hybrid" approach is amenable to both an accelerated cerebral cortical organoid protocol as well as an alternative long-term differentiation protocol. In addition to establishing a hybrid technical format, this protocol also offers phenotypic and morphological characterization of stage-specific cellular profiles using antibodies and fluorescent-based dyes for live cell imaging. © 2024 Wiley Periodicals LLC. Basic Protocol 1: hiPSC-based 2D monolayer specification into neural stem cells (NSCs) Basic Protocol 2: Serial passaging and 2D monolayer expansion of neuroepithelial progenitor cells (NPCs) Support Protocol 1: Direct cryopreservation and rapid thawing of NSCs and NPCs Basic Protocol 3: Bulk aggregation of 3D neurospheres and accelerated cerebral cortical organoid differentiation Alternate Protocol 1: Bulk aggregation of 3D neurospheres and long-term cerebral cortical organoid differentiation Support Protocol 2: High-throughput 3D neurosphere formation and 2D neurosphere migration assay Support Protocol 3: LIVE/DEAD stain cell imaging assay of 3D neurospheres Support Protocol 4: NeuroFluor NeuO live cell dye for 3D cerebral cortical organoids.

In the last decade, micro-RNAs (miRNAs) have emerged as major regulators of cell fate. They are involved in fine-tuning gene expression in normal developing tissues and are often aberrantly expressed in different disease states, including cancer. miRNAs are 20-25 nucleotide non-coding RNAs that repress the translation and stability of a large number of target mRNAs.
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\nThe study by Brett et al in the previous issue of AGING adds to our understanding of how miRNAs regulate the differentiation of adult neural stem cells (NSCs) [1]. The authors used primary cultures of neural stem/progenitor cells (NSPCs) isolated from adult mice to investigate the importance of a specific miRNA cluster, miR-106b~25, in regulating the proliferative potential and differentiation of NSCs. This miRNA cluster is located within an intronic region of the Mcm7 gene and codes for three different miRNA species, miR-106b, miR-93 and miR-25. Interestingly, activation of this miRNA cluster has been observed in different tumour types and is involved in the inhibition of anti-proliferative and pro-apoptotic genes, such as p21, Bim and TGF-beta [2,3]. Furthermore, this cluster is overexpressed in prostate cancer where it is involved in the downregulation of PTEN expression and also cooperates with its host gene Mcm7 to drive tumourigenesis [4].

Stem cells and cloning: The fuss, revisited.

The idea of being able to replace, restore, or regenerate damaged or diseased tissues and organs in order to establish normal function in the human body has never been so prominent and possible than with the development of the field of regenerative medicine. Using material from the body or activating the body’s own repair mechanisms to prepare and grow or heal irreparable tissues or organs is now a real possibility, as demonstrated by research and clinical studies published over the last 10 years. Born from the collaboration of several biomedical disciplines and clinical approaches, regenerative medicine promises the real possibility of not only replacing pharmaceutical solutions to disease but also paves the road to a more autologous and holistic approach in clinical treatment of disease and disease control. Historically, the foundation of regenerative medicine was cemented with the successful transplantation of corneas, soft tissue, and bone in the early twentieth century. The first kidney transplantation in 1954 was followed by pancreas, liver, and heart transplants in the 1960s and success continued in the 1980s with heart– lung and living donor liver and lung transplants. Progressively, an increase in demand for tissues and organs and subsequent decrease in organ availability has left a need for new technology to meet the demand for suitable organs and organ donors as well as leading to an increase in the disgraceful enterprise of black market organs. Currently, tissueengineered skin used for burn survivors and diabetic ulcers, products derived from tissue engineering to induce bone growth and regeneration, as well as the autologous reintroduction of ex-vivo engineered bladder are a reality, once again establishing a firm foothold for the development of further and more advanced applications for regenerative medicine. The discovery of stem cells by the Russian histologist Alexander Maksimov in 1908, as part of his theory of hematopoiesis followed by Joseph Altman and Gopal Das in the 1960s presenting evidence of constant stem cell activity in the brain and the demonstration of self-renewing cells found in the bone marrow of mice by James E. Till and Ernest A. McCulloch in 1963, established stem cell research and the development of stem cell therapy as a new and very promising and exciting discipline for regenerative medicine. Embryonic and adult stem cells are available from a variety of sources and hold variable differentiation potential, not to mention the myriad potential therapeutic applications, albeit associated with some ethical and political concerns. Mammalian stem cells can be sourced from either the embryo or the adult organism. Embryonic stem cells originate from the inner cell mass of blastocysts, whereas adult stem and progenitor cells can be found in various tissues repairing or replenishing adult tissue. Of specific interest and application in regenerative medicine are autologous adult stem cells accessible from bone marrow, adipose tissue, or blood. Contemporary stem cell therapy includes bone marrow transplants for the treatment of leukemia, whereas extensive current research is undertaken for the implementation of stem cell therapy for the treatment of cancer, spinal cord injuries, and Parkinson’s disease, as well as multiple sclerosis and muscle conditions including heart disease. The use of vascular grafts for heart bypass surgery and cardiovascular disease treatment have been researched extensively and are at the pre-clinical trial stage. Advances in therapeutic applications do not come without much debate and controversy surrounding risks associated with it. The risk that transplanted stem cells could form tumors and metastasize uncontrollably is but one of the concerns of stem cell therapy. Inducing forced expression of specific genes in order to derive pluripotent stem cells from non-pluripotent cells generate the so-called induced pluripotent stem cell (iPSC). However, significant risk is associated with reprogramming of adult cells to obtain iPSCs. Potentially, this risk could limit their use in patients. The use of viruses to genomically alter the cells may lead to the expression of cancer-causing oncogenes. Low-level laser therapy (LLLT) has been scientifically proven as a beneficial therapeutic modality for numerous diseases and diseased conditions. Using very specific laser and light-emitting diode irradiation parameters, specific cellular activities can be induced, namely, cellular proliferation and viability while stimulating mitochondrial activity, thereby increasing adenosine triphosphate (ATP) production, synthesis of DNA and RNA, and activating cellsignaling cascades including the production of reactive oxygen species (ROS), nitric oxide (NO) release, activating cytochrome c oxidase, and modifying intracellular organelle membrane activity, calcium flux, and expression of stress proteins. The molecular mechanism underlying these cellular activities is less well understood, and several research groups are conducting intensive research studies in an effort to elucidate the relation between these biological

Human Embryonic Stem Cells . Edited by J. Odorico, S. Zhang and R. Pedersen . (Pp. xxii + 391 , illustrated , ISBN 18599 62785 , £80 hardback.) Abingdon, UK : BIOS Scientific/Garland Science . It is now nearly seven years since the first description of human embryonic stem (ES) cell lines was published by Jamie Thomson. That event, closely apposed to the reports of the cloning of sheep (‘Dolly’), and then mice, by somatic nuclear transfer, catalysed substantial interest in the idea of regenerative medicine and the possibility that stem cells of all sorts might be harnessed for the replacement of tissues lost to accident or disease. At about the same time, a number of papers appeared, reporting that various somatic stem cells – which were previously thought to be committed to lineages from within their tissue of origin – might indeed exhibit substantial plasticity and be able to generate terminally differentiated cells corresponding to a much wider range of cell types. Thus, the current era of stem cell biology was born, though the concept of stem cells and their application in medicine is much older. One consequence of this resurgent interest in stem cell biology has been the publishing of several multi-author volumes devoted to stem cells and their potential. The volume Human Embryonic Stem Cells, edited by J. Odorico, S. Zhang and R. Pedersen, is the latest in a series produced by different publishing houses over the past few years. Paradoxically, however, as Jamie Thomson points out in his foreword to the present volume, progress in the field has been slow over this time. Prospects for adult stem cells have been dimmed by controversy over the phenomenon of ‘plasticity’, with the suggestion that at least some of the reported cases might be artefactual, depending upon cell fusion or rare transdifferentiation events. At the same time, progress with human ES cells was initially hindered by the difficulty of access to established lines, and the complexity of maintaining and expanding the cultures when they could be obtained. Although mouse ES cells have been available for over 20 years, they have mostly been used as tools to produce transgenic mice. With notable exceptions, few have investigated their cell biology for its own sake, and so there has been relatively little experience from studies of mouse ES cells to guide the study of their human counterparts. The development of ES cell lines was a systematic progression from the study of teratocarcinomas in the 1970s, by those who thought that these tumours might provide key insights into the mechanisms of embryonic development. It is then perhaps ironic that clues about how to control the behaviour of human ES cells in culture are now being provided by our detailed knowledge of developmental genetics, especially of the mouse. However, the human ES cell field is changing. Many laboratories in many countries have now derived human ES cell lines. In the current International Stem Cell Initiative, a collaborative venture to compare the properties of human ES cell lines derived worldwide, 75 independent lines derived in 17 laboratories and ten countries have been enrolled in the study. Meanwhile, reports are now beginning to appear in prominent journals, addressing the molecular mechanisms of human ES cell proliferation and differentiation – not merely describing their characteristics and potential for diverse differentiation. The present volume, unlike many of its competitors, focuses explicitly on human ES cells, and may be thought to mark the end of the first phase of human ES cell research. It brings together a series of authors who have contributed significantly to the field and it balances chapters discussing the basic biology of ES cells with others discussing their differentiation along specific lineages and eventual application. Despite the focus on ES cells, two early chapters provide useful comparative reviews of recent studies of adult stem cell plasticity and of mesenchymal stem cells. Further chapters address potential issues that will need to be addressed as derivatives of human ES cells are developed for eventual clinical application. Included here are two interesting and useful reviews of the ethical, legal and intellectual property aspects of human ES cell research and application. Undoubtedly, research in the new field of human ES cell biology is beginning to advance rapidly, so that it might be considered that the present volume will become rapidly obsolete. However, its well-organized and well-written chapters provide a valuable reference to the current state of the field for newcomers and established human ES cell researchers alike. Although our understanding of molecular mechanisms and the means of manipulating human ES cells is likely to change significantly in the future, much of what is contained in this volume will remain fundamental, and so of value to those working in this exciting new area of research for quite a few years to come.

Generating CNS Neurons from Embryonic, Fetal, and Adult Stem Cells

Embryonic stem cells are pluripotent and capable of unlimited self-renewal. Elucidation of the underlying molecular mechanism may contribute to the advancement of cell-based regenerative medicine. In the present work, we performed a large scale analysis of the phosphoproteome in mouse embryonic stem (mES) cells. Using multiplex strategies, we detected 4581 proteins and 3970 high confidence distinct phosphosites in 1642 phosphoproteins. Notably, 22 prominent phosphorylated stem cell marker proteins with 39 novel phosphosites were identified for the first time by mass spectrometry, including phosphorylation sites in NANOG (Ser-65) and RE1 silencing transcription factor (Ser-950 and Thr-953). Quantitative profiles of NANOG peptides obtained during the differentiation of mES cells revealed that the abundance of phosphopeptides and non-phosphopeptides decreased with different trends. To our knowledge, this study presents the largest global characterization of phosphorylation in mES cells. Compared with a study of ultimately differentiated tissue cells, a bioinformatics analysis of the phosphorylation data set revealed a consistent phosphorylation motif in human and mouse ES cells. Moreover, investigations into phosphorylation conservation suggested that phosphoproteins were more conserved in the undifferentiated ES cell state than in the ultimately differentiated tissue cell state. However, the opposite conclusion was drawn from this conservation comparison with phosphosites. Overall, this work provides an overview of phosphorylation in mES cells and is a valuable resource for the future understanding of basic biology in mES cells.

Neurodegenerative diseases are incurable and debilitating conditions characterized by the deterioration of brain function. Most brain disease models rely on human post-mortem brain tissue, non-human primate tissue, or in vitro two-dimensional (2D) experiments. Resource limitations and the complexity of the human brain are some of the reasons that make suitable human neurodegenerative disease models inaccessible. However, recently developed three-dimensional (3D) brain organoids derived from pluripotent stem cells (PSCs), including embryonic stem cells and induced PSCs, may provide suitable models for the study of the pathological features of neurodegenerative diseases. In this review, we provide an overview of existing 3D brain organoid models and discuss recent advances in organoid technology that have increased our understanding of brain development. Moreover, we explain how 3D organoid models recapitulate aspects of specific neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, and Huntington's disease, and explore the utility of these models, for therapeutic applications.

Pluripotent cells have the potential to differentiate into all of the cell types of an animal. This unique cell state is governed by an interconnected network of transcription factors. Among these, Oct4 plays an essential role both in the development of pluripotent cells in the embryo and in the self-renewal of its in vitro counterpart, embryonic stem (ES) cells. Furthermore, Oct4 is one of the four Yamanaka factors and its overexpression alone can generate induced pluripotent stem (iPS) cells. Recent reports underscore Oct4 as an essential regulator of opposing cell state transitions, such as pluripotency establishment and differentiation into embryonic germ lineages. Here we discuss these recent studies and the potential mechanisms underlying these contrasting functions of Oct4.

Brain organoids have become a valuable tool for studying human brain development, disease modeling, and drug testing. However, generating brain organoids with mature neurons is time-intensive and often incomplete, limiting their utility in studying age-related neurodegenerative diseases such as Alzheimer's disease (AD). Here, we report the generation of 3D brain organoids from human fibroblasts through direct reprogramming, with simplicity, efficiency, and reduced variability. We also demonstrate that induced brain organoids from APOE ε4 AD patient fibroblasts capture some disease-specific features and pathologies associated with APOE ε4 AD. Moreover, APOE ε4-induced brain organoids with mutant APP overexpression faithfully recapitulate the acceleration of AD-related pathologies, providing a more physiologically relevant and patient-specific model of familial AD. Importantly, transcriptome analysis reveals that gene sets specific to APOE ε4 patient-induced brain organoids are highly similar to those of APOE ε4 post-mortem AD brains. Overall, induced brain organoids from direct reprogramming offer a promising approach for more efficient and controlled studies of neurodegenerative disease modeling.