The impact of neural development research on understanding neural stem cells and human nervous system development

When I took my first neuroanatomy course in the mid-1970s with first-year dental and medical students, we learned the structures of the central and peripheral nervous systems (CNS and PNS) through memorization of their names and locations. Lectures on the development of the nervous system contained rather superficial information and constituted a tiny section of the course: the neural plate folds up into a neural tube, it then forms bulges that approximate neuroanatomical landmarks of the forebrain, midbrain, hindbrain and spinal cord, and some cells migrate away from the neural tube to form the sensory and autonomic ganglia. This lack of detail on the cellular processes by which neural structures develop was in part due to a lack of mechanistic understanding of how these events occur. Fortunately, over the next several decades, significant strides in research into how the normal nervous system develops were made that began to unravel the mysteries of the molecules and the cellular/tissue interactions by which the neural plate is induced from embryonic ectoderm, cause it to fold into a tube and regionalize, produce neurons and glia that migrate to their ultimate positions, direct axons to navigate through a complex and changing environment to reach their targets, and specify numerous distinct cell types, such as motor neurons, sensory neurons, interneurons, sensory receptors, astrocytes, and oligodendrocytes.1, 2 These discoveries came on the wave of new technologies being applied to embryos of a wide variety of animals—primarily chick, frog, fish, and mouse—that could be raised in the laboratory and were accessible to experimental manipulations and observation during the earliest phases of neural development. For example, in the 1980s, injections of vital dyes into single cells illustrated how axons grow to their targets, immature neurons navigate to their final locations, and lineage relationships specify different types of neural cells.3, 4 Monoclonal antibody technologies made it possible to identify the cell surface and extracellular matrix molecules that the migrating cells and axons use to navigate their environment and the unique proteins for different types of neurons and glia and their precursors.5-7 In the 1990s, the application of molecular cloning techniques, often based on the identification of pattern formation and neural identity genes discovered in Drosophila,8 led to the identification of the homologous genes in vertebrates that are responsible for the induction of the neural ectoderm, the axial segmentation of the neural tube, axon guidance, and neural cell identity.9-12 For the past 40+ years, there has been an explosion of information about how nearly every area of the CNS and PNS develops—including the molecular signatures of neural stem and progenitor cells and their numerous neuronal and glia progeny.13 As these exciting findings were accumulating, developmental neurobiologists—such as myself—who were teaching the next generation of health professionals and biomedical students tried to incorporate them into their first-year human neuroanatomy courses, but it was difficult to do justice to these topics. There were time constraints in the curriculum and not all students were enthusiastic about learning information that did not come from human material. Traditionally, human neuroembryology has been limited to descriptions of tissue from embryos and fetuses that had been lost during pregnancy, with no opportunity for experimental interrogation of mechanisms. While it is assumed that most of what has been discovered in the animal models is relevant to human nervous system development, until recently, this could not be experimentally tested. Teaching the exciting molecular mechanisms by which the vertebrate CNS and PNS form thus has been confined mostly to upper level courses for research-oriented graduate students and neurology/neurosurgery residents. Therefore, the current practice in teaching neural development to health professional students has not advanced very much since when I was a graduate student. Perhaps a segment of a lecture might be devoted to the developmental cause of a congenital syndrome—such as the role of the sonic hedgehog gene in holoprosenecephy—might be mentioned in a clinical correlation session, but in my experience, developmental mechanisms receive little attention. However, there are major changes afoot with the recent availability of human-derived embryonic stem cells (ESCs), induced pluripotent stem cells (IPSCs), and their in vitro assembly into composite structures called organoids and assembloids.14, 15 These human models of neural development have been made possible by the decades of research in animal model systems that discovered the transcription factors, signaling pathways, epigenetic modifications, and cell/tissue/extracellular matrix interactions required for normal CNS and PNS development. How has this come about? It has long been known that many tissues in the body, such as the cornea, skin, muscle, and blood, contain stem cells that are called upon to correct damage from disease or trauma. However, the adult mammalian nervous system was not thought to contain stem cells that could give rise to neurons, thus prohibiting regeneration after damage. However, the development in the 1960s of a technique called “birth-dating,” which identifies cells undergoing mitosis, provided the first evidence of a low level of neurogenesis in the adult brain. After decades of experiments and debate, it is now firmly established that there are niches in the adult brains of many vertebrates—including human—that contain stem and progenitor cells that generate both neurons and glia.16 In the forebrain, these are confined to the subventricular zone of the lateral ventricles and the subgranular zone of the hippocampus. The new neurons generated in the hippocampus are thought to play an important role in learning and memory, and the progeny of a subset of neural stem cells generated in the subventricular zone migrate from the lateral ventricles into the olfactory bulbs to become new interneurons.17, 18 Confirmation of adult neurogenesis gave hope to the neuroscience research community that these cells could be used to replace neurons lost due to trauma, stroke, or neurodegenerative disease. This led to an explosion in research, again in animal model systems, to identify the molecular signatures of neural stem and progenitor cells in the niches of the adult brain and the signaling pathways that specify different cell types. The hope was to apply this basic knowledge to cultured cells to differentiate the needed neuronal type, for example, midbrain dopamine (DA) neurons for Parkinson's, GABA neurons for Huntington's, motor neurons for Amyotrophic Lateral Sclerosis and Spinal Muscular Atrophy, and photoreceptors for macular degeneration. One obstacle to harvesting endogenous neural stem cells for clinical applications, however, was the fact that there are only a very few endogenous pluripotent cells available in the brain of an individual, and they are a heterogeneous mixture of stem, progenitor, and supporting cells.19, 20 This caveat led to the development of approaches to transform cultured ESCs, derived from the blastocyst stage, and IPSCs, derived from adult blood cells or fibroblasts, into neural tissue. In culture, one could grow large numbers of these cells and use the rich understanding of the cellular and molecular processes that drive neuronal differentiation in the embryo/fetus derived from animal model research to engineer culture conditions to produce the desired cell types (e.g., DA neurons for Parkinson's).21 However, there were problems to be solved with this new approach: because the cultures usually contained at least a few stem cells that maintained their ability to proliferate, tumors often formed; cells that differentiated in culture often did not functionally incorporate into existing brain circuits after transplantation; two-dimensional cultures tended to be difficult to maintain for the long time needed to obtain human mature neurons; and the availability of human ESC lines was limited. Finally, although work in animal models had yielded fundamental insights into neural development, there are some issues that are uniquely human, including a longer developmental time frame and a greatly expanded cerebral cortex.15, 22 An initial breakthrough was figuring out how to grow ESCs in three-dimensions (3D), which allowed cells to interact in a more natural environment. It was found that by sequentially treating 3D cultures with the factors known in the embryo to induce and pattern the nervous system (based on basic neural developmental research in animal models), the tissue could self-assemble into a structure reminiscent of a specific brain region. One of the most striking examples was the in vitro formation of an optic cup.23 In an effort to apply the new knowledge of neural development to disease treatment, many studies attempted to work out a clinical treatment of various neurodegenerative diseases. For example, it was known that patients with Parkinson's disease suffered from the degeneration of DA neurons in the substantia nigra, and its symptoms could be ameliorated by the administration of Levodopa (l-Dopa), a precursor of DA. However, certain side effects of the drug regime rendered this a short-term treatment. Thus, researchers sought an approach that would replace the lost DA neurons themselves, not just the neurotransmitter. Initial therapies included transplanting pieces of ventral mesencephalon, which is the site of the developing substantia nigra, from human fetuses that had been lost during pregnancy into the striatal region of adult patients. While there was success for some patients, the material was very limited, its use raised ethical concerns, patients had to be on long-term immunosuppressant therapy, and because the grafts contained dividing stem/progenitor cells, tumors often formed.24 As an alternative, recent techniques have been designed to culture human IPSCs to the point of being specific types of progenitor cells that are then sorted to remove the dividing stem cells to avoid tumor formation. A recent successful example transplanted DA progenitor cells, derived from human IPSC lines, into the putamen of Parkinson patients; the cells survived, differentiated into DA-producing neurons, did not form tumors, and resulted in mild improvements in motor function in 4/6 patients.24 Of course, long-term efficacy trials are still needed, but this is an encouraging application of basic neural development research to stem cell technology to address a significant neurological problem. Another recent advance that has truly excited the clinical and basic science communities is the development of “organoids” and “assembloids” from human IPSCs.14, 15 Improving upon the techniques mentioned above to produce 3D cultures, clusters of cells have been shown to assemble into multilayered brain-like structures—hence, the name “organoid.” These have been used, for example, to study (1) how human neurons migrate from the regions where they undergo terminal mitosis to their final adult location; (2) the pattern of gene expression that leads to the differentiation of a specific cell type in different brain regions; (3) what molecular signatures are disrupted when human IPSCs are derived from patients with a particular neurological disease; and (4) whether the genes linked to conditions such as autism, schizophrenia, and Alzheimer's alter normal brain development and function. Organoids have the advantage of eliminating the ethical concerns about destroying human blastocysts to make ESCs or depending on aborted fetal material for grafts. Research over the last decade has produced an impressive array of human organoids that closely resemble, in architecture and molecular signatures, several different brain regions.14, 15 These have been very useful to determine which aspects of neural development studied in animal models are conserved in humans and which are human-specific.22 The next logical step has been to put organoids derived from different brain regions in close apposition in the culture dish to study how functional neural networks are built; these composite organoids are called assembloids.14 Assembloids have been used to study how circuit-forming events are disrupted in certain neurological syndromes, to understand how motor cortex activity triggers muscle contraction and how pain is processed from the PNS to the sensory cortex. In addition to studying processes that are disrupted in various neurological syndromes by using patient-derived IPSCs, organoids and assembloids are making it possible to test drugs for their ability to ameliorate the cellular dysfunctions in these diseases. Because these cells are readily available compared to endogenous neural stem cells, fetal graft material, and human ESC lines, protocols are scalable to produce sufficient material to carry out clinical trials with statistical power. Of course, for the long-desired therapies to become reality, the best practices for these new technologies must be standardized to reduce lab-to-lab and culture-to-culture variability.25 How will the new approaches for studying neural stem and progenitor cells in a culture dish impact our understanding of the development of the human nervous system? First and foremost, because they make the human nervous system available for rigorous, experiment-based research to define the normal mechanisms that form the CNS and PNS, they will allow the decades of essential information gleaned from animal models to be validated in a human cell culture system. In turn, the results from these experiments are likely to impact the extent to which principles of neural development are included in the curricula of health professional students. Topics that are likely to be relevant in the near future include those developmental processes found to be of clinical relevance and with the potential for new therapeutic approaches; the use of assembloids derived from healthy individuals' IPSCs to reveal relationships between different brain regions that have not been apparent from the traditional techniques used to build the current dogma regarding neuroanatomical and neurophysiological relationships; and the use of assembloids derived from patients' IPSCs to reveal aberrant interactions between neural cells that underlie neurological congenital syndromes and disease.15, 22 Thus, the maturation of these technologies is anticipated to play a major role in our understanding of many human neural developmental and neurological processes. Sally A. Moody: Conceptualization; writing – original draft. This work was not funded by an extramural agency. The author claims no conflicts of interest. This work contains only literature citations, no original data. Sally A. Moody received her MS in Anatomy from the University of Maryland and her PhD in Neuroscience from the University of Florida. She was a postdoctoral fellow with Marcus Jacobson the Neurobiology Department at the University of Utah. She was Assistant, then Associate, Professor of Anatomy and Cell Biology and of Neuroscience at the University of Virginia, and moved her laboratory to the Geirge Washington University where she served as Professor and then Chair in the Anatomy and Cell Biology Department.