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Perspective Neural Stem Cells: Generating and Regenerating the Brain Fred H. Gage1,* and Sally Temple2 1Laboratory
of Genetics, The Salk Institute for Biological Studies, 10010 N. Torrey Pines Road, La Jolla, CA 92037, USA Stem Cell Institute, One Discovery Drive, Rensselaer, NY 12144, USA *Correspondence: [email protected] http://dx.doi.org/10.1016/j.neuron.2013.10.037 2Neural
One of the landmark events of the past 25 years in neuroscience research was the establishment of neural stem cells (NSCs) as a life-long source of neurons and glia, a concept that shattered the dogma that the nervous system lacked regenerative power. Stem cells afford the plasticity to generate, repair, and change nervous system function. Combined with reprogramming technology, human somatic cell-derived NSCs and their progeny can model neurological diseases with improved accuracy. As technology advances, we anticipate further important discoveries and novel therapies based on the knowledge and application of these powerful cells. Introduction It is an understatement to say that the conceptual advances and practical applications of stem cell research have been game changers for the field of neuroscience. When the term ‘‘stem cell’’ was used 25 years ago, the context was typically adult tissue homeostasis, mouse genetics, or early development. Thanks to several landmark advances in the area of developmental neurobiology, neural stem cells (NSCs) are now central to our discussion of how the brain forms. While there was evidence for adult neurogenesis early on, it was controversial, and the underlying cells responsible for continued neuronal and glial production were not characterized. Today it would be difficult to consider adult neurogenesis without reference to endogenous NSCs and their niches. Although early researchers had determined that individual transcription factors directed cell fate, as in MyoD for muscle, and had done pioneering experiments proving that oocyte proteins could dedifferentiate a somatic cell nucleus, they could not have imagined the explosion of reprogramming that now allows us to generate human neural cells from induced stem cells and enables us to model nervous system diseases in entirely new ways. Progress at the basic research level has also been astonishing, and we are already witnessing the translation of NSC science, with several clinical trials ongoing and more in the planning stages. In the following Perspective, we will review some of the milestones of the last 25 years in NSC research. Rather than providing a comprehensive review of these advances, we intend to highlight the major events and discoveries that we feel have made the most important contributions to our field. In particular, we will focus on the shifts in the field around the concept of adult neurogenesis and stem cells in the adult brain, especially in the hippocampus. We will discuss the more recent development of methodologies for reprogramming and induced pluripotent stem cells (iPSCs) and outline our views on the promise of NSC-based approaches for the treatment of disease. Significant milestone advances that have driven NSC research forward have been summarized in Table 1, and we have also provided a tools wish list that would enable researchers to address some key 588 Neuron 80, October 30, 2013 ª2013 Elsevier Inc.
remaining questions concerning NSC biology (Table 2). The views here represent our personal perspectives on what has been particularly significant; we readily acknowledge that this only reflects a fraction of the interesting and important work in the field, and we apologize to those whose work we have not had space to discuss and reference. As you read this Perspective, we hope to inspire you to imagine the conceptual advances and new applications of NSC research over the next 25 years. I. Early Halcyon Days for Studies on the Development of the Nervous System It is difficult to imagine how much in the dark we were about mammalian nervous system development back in the 1980s. One of the burning questions at that time was whether or not common progenitor cells for neurons and glia even existed. Stem cells were not generally considered a part of brain development but rather the building blocks of other, more plastic tissues. The tools available to us to address these fundamental questions were limited. In vivo studies of the developing brain relied heavily on histological stains that would have been familiar to Camillo Golgi, in vitro culture media were primitive, growth factors to stimulate division of neural progenitors had not been identified, and there were only a handful of antibodies to identify neural cell types. But we were young researchers in a young field, excited by the questions, undaunted by the challenges, and fortunate to be on hand at the beginning of arguably the most extraordinarily productive quarter century in developmental neuroscience. Identifying the Progeny of Single Cells Revealed Heterogeneous Progenitor Cells, Including NSCs As is often the case in developmental studies, invertebrate research had taken the lead, and methods to genetically mark individual progenitor cells in vivo revealed unexpected clonal boundaries and patterns that revolutionized our comprehension of how insects are built. With this inspiration, the question was how to look into the developing CNS of large, cytologically complex, opaque organisms that inconveniently developed in utero. The door was opened by the development of retroviral
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Perspective lineage-tracing methods applicable to mammals. In the mouse cerebral cortex, for example, these techniques showed that most progenitor cells at midgestation generated surprisingly small clones, typically solely of neurons, that could migrate quite widely (Price and Thurlow, 1988; Turner and Cepko, 1987; Walsh and Cepko, 1992). Earlier infected clones could span multiple cortical layers, indicating multipotency for neuronal production (Kornack and Rakic, 1995). We learned that mixed neuron-glia clones were rare in the cortex but far more numerous in the spinal cord, where clones containing motor neurons, interneurons, and glial progeny were observed (Leber and Sanes, 1995). To determine whether the diversity of clones seen in vivo reflected environmental cues that the clonal progeny encountered or cell-intrinsic heterogeneity, researchers began to perform clonal studies in vitro, observing single embryonic cells growing in a standardized environment to uncover cell-based differences. Most embryonic progenitors produced clones of solely neurons, others of solely glia, but, most excitingly, a small subset of progenitors had large proliferative potential and incontrovertibly produced both neuronal and glial progeny. These progenitors also demonstrated some capacity for self-renewal, a cardinal property of stem cells (Anderson, 1989; Cattaneo and McKay, 1990; Davis and Temple, 1994; Kilpatrick and Bartlett, 1993; Temple, 1989). These discoveries led to the concept that nervous system development, both peripheral and central, was similar to that of the blood system and relied on fundamental, multipotent NSCs that repeatedly produced more restricted neural progenitors that, in turn, divided just a few times to produce small numbers of differentiated progeny. Today, while studies of progenitor heterogeneity are advancing—for example, by the identification, within germinal zones, of cells with diverse morphologies and diverse markers (Ayoub et al., 2011; Fietz et al., 2012; Franco et al., 2012; Kawaguchi et al., 2008; Pinto et al., 2008)—embryonic neural progenitor cells (NPCs) are still classified into just two basic types: multipotent NSCs and transit amplifying/intermediate progenitor cells (IPCs) (Englund et al., 2005). We have yet to define most of the progenitor subsets contributing the vast array of fates seen clonally in vivo and in vitro and to understand their key regulators and their role in neural development. These will be significant goals for the next decade. Uncovering the Dynamics of NSC Behavior Development can be thought of as increasing cellular complexity over time. We marvel at the precise orchestration of cell proliferation and then differentiation into innumerable types of neurons and then glia. How are these events choreographed? Pioneering heterochronic transplantation studies demonstrated that early progenitors have a wide multipotency but late progenitors are unable to produce the earlier fates (Frantz and McConnell, 1996; McConnell and Kaznowski, 1991). This finding led to a key idea that the potential of CNS stem cells is progressively, temporally restricted. How do CNS progenitor cells change over time? The development of tools to record extended time-lapse movies of CNS germinal cells ex vivo has yielded enormous insights. Movies of isolated cortical clones growing in 2D cultures showed that the lineage trees of isolated murine CNS progenitor cells were highly reminiscent of those of invertebrates and, astonishingly,
that individual cells were programmed to recapitulate the timing of diverse progeny seen in vivo, including their gradual restriction in potency (Shen et al., 2006). Combining retroviral labeling and slice culture, we could observe cortical progenitor cells in a system that retained much of the normal 3D niche architecture. This technique revealed that radial glial cells (RGCs), which span from their soma in the ventricular zone (VZ) to the pial surface, were the fundamental progenitor cells for neurons (Noctor et al., 2001) and later glia. Combined with in vitro studies using transgenic reporters for RGCs (Malatesta et al., 2000), this finding led to the notion that embryonic multipotent CNS NSCs were a subset of RGCs. The advancement of sophisticated imaging techniques and analytical tools (Winter et al., 2011) has great potential to further illuminate progenitor cell behavior over time. We look forward to observing multiple signals simultaneously, enabling us to follow the expression and movements of not only single genes or proteins but also of pathways and networks, as the progenitor cells change during neural development and after challenges. Much progress has been made to understand the temporal control of NSC output. Steps in the timing process rely on production of gliogenic cues, such as cardiotropin, transcription factor sequences, DNA methylation changes, and chromatin modifications (Barnabe´-Heider et al., 2005; Hirabayashi et al., 2009; Namihira et al., 2009; Pereira et al., 2010). Yet today, key aspects of the mechanisms that underlie progenitor temporal control remain enigmatic, presenting a challenge that is somewhat ahead of the tools currently available. We need the means to rapidly record dynamic changes in multigene expression, transcription factor binding, and chromatin structure and, ideally, to do so within living progenitor cells as they divide and move down the lineage trees. It is a lot to ask but, given the rapid evolution of single-cell tools, we might get there sooner than expected. How Are CNS Stem Cells Regionally Patterned? One of the central discoveries in developmental neuroscience that has emerged in this past 25-year era concerns how the nervous system is regionally patterned. Embryological manipulations—first in chick, then with transgenic mice—elucidated the morphogenic gradients that pattern neural tissue, for example, ensuring that motor neurons and oligodendrocytes arise ventrally and interneurons arise dorsally in the spinal cord (Briscoe et al., 1999; Liem et al., 1997). Other notable studies revealed that specific CNS regions can be organizers; for example, the midhindbrain isthmus drives midbrain patterning via release of FGF8, so that implanted beads containing FGF8 cause duplication of the cerebellum (Martinez et al., 1999). Studies of mouse mutants that were almost perfect apart from the lack of specific brain regions showed that the CNS develops as modules defined by transcription factor domains (Puelles and Rubenstein, 2003). One fascinating question that we have yet to answer is how morphogenic gradients intersect with and activate specific lineage programs in NSCs and their progeny, so that discrete, regionally appropriate progeny are made. While CNS development is modular, cells can cross regional boundaries. In a landmark demonstration, GABAergic neurons in the forebrain were shown to be born ventrally and migrate into the overlying dorsal cortex (Anderson et al., 1997). This Neuron 80, October 30, 2013 ª2013 Elsevier Inc. 589
Proliferation markers: tritiated cell labeling, Brdu, Edu, Ki67
Neural cell-type-specific antibodies
In situ hybridization of mRNAs in CNS germinal zones
Viral vectors for lineage tracing and gene manipulation
Single-cell cloning for CNS neuronal and glial progenitor cells
Confocal microscopy
Surface marker definition and cell separation
Serum-free culture methods with identified NSC growth factors
Long-term time-lapse microscopy
Conditional mutations in mice
Microarrays
RNA interference (microRNA, siRNA, shRNA)
hESCs SCNT
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1970s FACS; 1989 MACS
1990s
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Tools
1961
Approximate Impact Start Date
590 Neuron 80, October 30, 2013 ª2013 Elsevier Inc. How are neural progeny derived from human pluripotent cells?
How does gene knockdown in vitro and in vivo alter NSC behavior?
What genes are expressed in NSCs?
What is the impact of gene LOF or GOF on NSCs?
What is the identity of neuronal and glial progenitor cells in 3D slices?
Which factors stimulate NSC proliferation and differentiation in vivo?
What are the major subtypes of embryonic and adult progenitor cells?
RNAi library screening to identify NSC regulators
Gene expression after perturbations?
How are NSCs regionally patterned?
What changes occur during CNS germinal cell division?
Which factors expand progenitor types ex vivo?
How do prospectively isolated NSCs/NPCs behave?
3D tissue imaging
What is the relationship of NSC subtypes to brain tumor-initiating cells?
What are the lineage trees of individual NSCs and NPCs?
How do environmental factors impact NPC/NSC behavior?
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Production of RPE cells and other neural products for clinical trials
RNA interference therapeutics
Production of diverse neural progeny from NSCs and PSCs
Exogenous factors to enhance NSC activity
Enrichment of progenitor cells for transplantation
Pathology
Do self-renewing NSCs exist and in which CNS region?
What intrinsically different types of NSCs and NPCs exist? Identity of subcellular NSC markers in vivo
Targeted gene therapy
How do progenitors generate progeny in slice culture?
What progeny do single progenitors generate in vivo? Are NSCs functional?
Are there common progenitor cells for neurons and glia?
Pathology
How do NSCs respond to disease, drugs, and environmental perturbations?
Antibodies to select cells for transplant; drugs that impact stem cell growth, e.g., anti-angiogenic Abs
Assessment of cell growth after cell transplantation
Translation
What genes are expressed by NSCs, NPCs, and their progeny?
How do NSCs respond to disease, drugs, and environmental perturbations?
How is NSC proliferation regulated by environment?
What types of progeny do NSCs produce?
Do NSCs exist in the adult?
How are germinal niches structured in vivo?
What is the birthdate of neural cells during development?
Questions
Table 1. Milestones in NSC Research Related to Technology Development and Application
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Perspective
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Gene repair; creation of appropriately matched control and disease lines
How is chromatin modified in NSCs and their progeny?
Can somatic cells be turned into cells resembling embryonic stem cells?
What is the RNA complement of NSCs and their progeny?
Can somatic cells be converted directly into neurons and NSCs?
Does specific gene alteration impact NSC behavior?
Chip-seq Gro-seq
Mouse, human iPSCs
RNA-seq Gro-seq
Direct reprogramming
TALEN and CRISPR gene editing
2005
2006–2007
2008
2010
2010–2013
What genes are critical to specify neural fates?
What is the functional effect of optogenetic engineering in NSCs? Optogenetics 2002
Transcriptome studies after perturbations
Questions Tools Approximate Impact Start Date
Table 1.
Continued
How do chromatin alterations alter NSC behavior?
Report gene expression more accurately
Rapid generation of purer cell populations
Disease-related changes in RNA expression
Neural disease modeling; drug screening; potential autologous transplantation
Diagnosis of chromatin changes in disease; development of drugs to target
To stimulate NSC progeny after transplantation
Translation
Perspective finding—that almost the entire inhibitory neuron complement of the cortex arose from NSCs that were born elsewhere—was most surprising. Migration was not just along radial glia but tangential (O’Rourke et al., 1995), and the routes of all sorts of peripatetic CNS progenitor cells have now been revealed, from the pioneering Cajal-Retsius neurons from the cortical hem (Bielle et al., 2005) to the vast spreading migrations of different waves of oligodendrocyte precursors (Kessaris et al., 2006; Timsit et al., 1995). Such mixing increases the richness of connective possibilities, and cell migratory defects will continue to be explored as the cause of multiple neurological disorders. Building the Foundation of Human NSC Studies Much of our understanding of mammalian neural development comes from mouse studies, and resources such as BGEM, Genepaint, the Allen Brain Atlas, MGI, and KOMP enable us to question further and deeper. Still, the mouse is lissencephalic, its neuronal complement is born in essentially 7 days, and no one doubts comparative studies that indicate significant differences in how the 1,000-fold larger human brain is built over 9 months of gestation (Zeng et al., 2012). But, thanks to vast improvements in tools and resources and to the advent of human stem cell biology, we are now ready to take on the task of understanding human CNS development. The next 25 years, we predict, will witness great strides in that area. Already, we know that the human forebrain has not just the VZ and subventricular zone (SVZ) but also a significantly expanded germinal zone, the outer SVZ, which helps account for the orders of magnitude increase in its size and complexity (Bystron et al., 2008; Hansen et al., 2010). Dissection of these germinal layers provides a clue to the key transcription factors and pathways that characterize their constituent cells (Fietz et al., 2012). In the future, fundamental molecular studies will expand our knowledge of the temporal patterns of gene expression and epigenomic changes that accompany human neural development (Kang et al., 2011). New techniques for creating in vitro human neural organoids with salient morphologic features such as retinal and cortical layering (Aoki et al., 2009; Eiraku et al., 2011; Lancaster et al., 2013; Meyer et al., 2011) will enable 3D imaging of how human CNS progenitor cells work and will broaden our understanding of CNS morphogenesis. Progress over the past 25 years in characterizing embryonic NSCs and understanding their patterning, lineages, and role in nervous system development has been and continues to be complemented by tremendous strides in the characterization of adult NSCs, enabling cross-fertilization of ideas and tools and encompassing adult learning and memory, environmental regulation, cancer, and aging. II. Discovery and Elaboration of Adult NSCs and Neurogenesis History: Cell Division to Neurogenesis in the Adult Brain The observations of cell division and differentiation in the adult brain emerged from studies of brain development and were greatly advanced by the early application by Leblond and colleagues of tritiated thymidine, which incorporates into the DNA of dividing cells and can be detected by autoradiography. Using this labeling technique, Leblond and colleagues observed and concluded that glial cells were probably dividing throughout Neuron 80, October 30, 2013 ª2013 Elsevier Inc. 591
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Perspective Table 2. Wish List of Technologies that Will Accelerate or Enable Important Directions in NSC Research Tool
Questions
Translation
More antibody and other markers to prospectively identify NSCs and NPCs
What is the range of types of NSCs and NPC present in mouse and human?
Understanding the contribution of NSC lineages to disease
Diverse fluorescent markers to report multiple genes, multiple proteins
Visualize complex elements in the same cell
Broad multiplex imaging: for immunostaining, in situ and gene expression reporting
To track multiple molecules simultaneously—analysis of networks, pathways and multimolecular complexes
How do NSC pathways change during development and with aging?
3D cultures that recapitulate human neural development; integrate engineering
What mechanisms underlie human NSC development into neural progeny?
What morphologic changes accompany CNS development?
Improved cell transplants and prostheses and disease models
More 2D and 3D imaging and analysis tools ex vivo
How do NSCs interact with niche cells, statically and dynamically?
Quantification of image information
Imaging standards for cell products; pathology assessment
High definition (deeper and higher resolution), in vivo imaging
What is the dynamic behavior of NSCs in vivo in the normal niche?
How do NSCs migrate and integrate in vivo?
Precise cell tracking after transplantation in vivo
Closer collaborations between basic, clinical, applied, and commercial stake holders
What are the clinical implications of NSC-based discoveries?
Critical for effective translation
Better nonviral transfection methods
Accelerate and reduce the cost of answering many questions about NSCs
Allow easy, high-throughput screening
Improved models of neurological disease
What is the disease mechanism?
the parenchyma (Smart and Leblond, 1961). They specifically found dividing cells in the subependymal zone (SEZ) but did not observe neurogenesis because the percursors born in the SEZ, later renamed the SVZ, must migrate to the olfactory bulb before they differentiate into neurons. Soon after these pioneering studies, Joe Altman, using the same techniques, observed dividing cells in the subgranular zone and speculated that neurogenesis occurred in the adult rat and cat dentate gyrus (DG) (Altman, 1962, 1963). Then, in 1965, he and Gopal Das provided the first strong evidence for neurogenesis in the adult brain (Altman and Das, 1965), reporting on the migration of cells that were born postnatally in the SVZ and matured into neurons in the olfactory bulb. In 1969, Altman was the first to describe the rostral migratory stream, located between the SVZ and olfactory bulb (Altman, 1969). Surprisingly, there was little follow-up to these discoveries for almost 10 years, when Kaplan and Hinds provided electron microscopic data, combined with tritiated thymidine staining, that showed the existence of adult-born neurons in the DG and the olfactory bulb (Kaplan and Hinds, 1977). The next phase in the early history of adult neurogenesis moved to the avian brain, where Goldman and Nottebohm first detected what they reported was neurogenesis in adult birds (Goldman and Nottebohm, 1983); Paton and Nottebohm then demonstrated functionality by unit recording and then autoradiography of thymidine-labeled neurons (Paton and Nottebohm, 1984). After another period of little activity in the area, four developments and discoveries changed the perception of neurogenesis in the mammalian brain in the 1990s. The first was the observation that proliferation levels of the early progenitor cells and subsequent numbers of newborn neurons were regulated. Gould, 592 Neuron 80, October 30, 2013 ª2013 Elsevier Inc.
What pathways are best targeted?
Could supplant animal models for efficacy and some tox studies
Cameron, and McEwen demonstrated that stress levels negatively affected the numbers of proliferating cells in the DG (Gould et al., 1992). This finding was followed by a series of observations demonstrating that neurogenesis could be substantially increased by running (van Praag et al., 1999), that housing animals even for short periods of enrichment in complex environments increased robustly the number of surviving newborn neurons (Kempermann et al., 1997), that learning itself could influence adult neurogenesis (Do¨bro¨ssy et al., 2003; Gould et al., 1997), and that antidepressant drugs (SSRIs) as well as alcohol (Nixon and Crews, 2002) could influence components of the adult neurogenesis process (Malberg et al., 2000). Around this same time, neurogenesis was shown to decrease with age but persist throughout life (Kuhn et al., 1996). A second development was the advancements in immunohistological techniques, combined with the application of confocal microscopy to the study of adult neurogenesis and, importantly, the application of stereological techniques for labeling dividing cells (in particular bromodeoxyuridene [BrdU]) and neurons (initially NeuN). These techniques allowed the simultaneous colabeling of neurons and proliferating cells and quantification of the changes in these cells in vivo, convincingly demonstrating that the dividing cells in the DG indeed became neurons (Kempermann et al., 1997; Kuhn et al., 1996, 1997). Using these techniques combined with transplantation, Lois and AlvarezBuylla demonstrated that endogenous and engrafted SVZ cells migrated into the olfactory bulb (Lois and Alvarez-Buylla, 1994). They also provided evidence for the surprising finding that stem cells in the adult SVZ expressed the astrocyte marker GFAP (Doetsch et al., 1999).
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Perspective The third important advance was the application of these newly applied techniques to identify new neurons in the DG of cancer patients who were given BrdU for diagnostic purposes (Eriksson et al., 1998), generalizing the findings of adult neurogenesis to humans. This demonstration of human adult neurogenesis was confirmed recently using carbon14 dating techniques on neurons derived from individuals born during the period of above-ground nuclear testing (Spalding et al., 2013). The fourth important development was the identification of methods to isolate, propagate, and differentiate progenitors from the adult CNS in defined culture conditions. This breakthrough was first achieved from dissections of the lateral wall of the striatum to obtain cells of the SVZ and then the expansion of the proliferating population into what came to be known as neurospheres (Kilpatrick and Bartlett, 1993; Reynolds and Weiss, 1992). The subgranular zone (SGZ) population of dividing cells was isolated from the hippocampus and then expanded in vitro and maintained as monolayers (Palmer et al., 1995, 1997). The ability to isolate, maintain, expand, and differentiate these precursor cells in vitro led to the ability to explore, in more detail, the cellular and molecular nature of the cells and the mechanisms that regulated their behavior. The in vitro cells could then be tested in vivo using the newly developed in vivo tools. The demonstration of neurogenesis in humans, along with its regulation by behavior and the environment, highlighted its relevance to the scientific community and helped motivate research into the wider regenerative potential of NSCs. The Niche Over the ensuing decade (2000–2010), many of the details of the phenomenon of neurogenesis were revealed. Importantly, the anatomical location and cellular constituents of the ‘‘niche’’ where NSCs are born and maintained were found to be more complex than anticipated but to be similar to niches that were being discovered for stem cells generated in other adult organs. The phenomenon of neurogenesis can be delineated into four processes: cell proliferation, migration, cell survival, and neuronal differentiation. Each aspect is critical to the overall levels of neurogenesis. For example, NPC proliferation occurs in other regions of the adult brain but NPCs do not differentiate into neurons there, either maintaining the properties of precursors or becoming glia. However, NPCs isolated from these nonneurogenic regions, such as cortex and optic nerve, in the adult brain retain the potential to become neurons in vitro when expanded in FGF-2 and treated with differentiating molecules like retinoic acid and Forskolin, indicating that extrinsic factors play a major role in stimulating NPCs to differentiate into neurons (Palmer et al., 1999). Additional support for the importance of the neurogenic microenvironment comes from the finding that NPCs located in the SVZ and SGZ are the only ones that adopt a neuronal cell fate under normal physiological conditions in the adult brain; however, if these NPCs are isolated from the SVZ or GVZ with the techniques described above and then transplanted into ectopic regions of the adult brain, they differentiate mostly to oligodendrocytes and astrocytes (Seidenfaden et al., 2006). In contrast, NPCs from the DG can differentiate into olfactory bulb neurons when grafted to the SVZ (Suhonen et al., 1996) and, more dramatically, NPCs isolated from a nonneurogenic re-
gion, such as the spinal cord, can differentiate into neurons when transplanted into the DG, supporting the idea that external cues from the local microenvironment promote the neuronal differentiation of NPCs (Shihabuddin et al., 2000). The SVZ and SGZ represent neurogenic niches or local microenvironments that permit and support neurogenesis. To date, many of the cellular constituents of the niche have been identified, including astrocytes (Song et al., 2002b), endothelial cells (Shen et al., 2004), microglia (Sierra et al., 2010), and the blood vascular system itself (Palmer et al., 2000). A more complete understanding of the molecules and events that regulate the niche and its influence on neural stem cell behavior is being revealed on a daily basis in the current literature. What will be very useful—and has not yet been achieved because of the optical limits—is the observation in real time of stem cells in their niche and the temporal process by which the cells interact with their microenvironment to generate neurons. Morphological Transition of Stem Cells to Neurons New neurons born in the adult brain undergo a maturation process that takes several months before they are essentially equivalent to mature neurons. Arising from a local stem cell population (Gage, 2000), adult-born neurons initially are not directly connected at all to local circuitry. Nonetheless, they are apparently responsive to local neurotransmitters, likely through spillover from nearby synapses (Song et al., 2002a). It takes about 2 months for newborn neurons to reach morphological maturity. Although no significant structural differences are observed between fully mature adult-born and perinatal-born neurons, the maturation process is delayed in the adult (Zhao et al., 2006), and it is very likely that this delay is crucial for their function both as young neurons and subsequently as mature neurons (Aimone et al., 2009). Notably, the spine formation process of adult-born neurons appears to be different from that of perinatal-born neurons in that adult-born neurons preferentially target pre-existing synapses; little is known about the underlying mechanisms (Toni et al., 2007). One hypothesis is that glutamate spillover may play a chemoattractive role and induce filopodia growth toward active synapses (Toni and Sultan, 2011; Toni et al., 2007). Local synaptic activity may induce glutamate release and activate glutamate receptors in filopodia, which induces new filopodia to target the existing synapse (Toni and Sultan, 2011). This structural difference parallels the differences between the physiologies of immature and mature granule cells. Newborn neurons display a high input resistance (Espo´sito et al., 2005), receive less inhibition (Li et al., 2012), and have been shown to exhibit considerably greater synaptic plasticity than mature granular neurons (Ge et al., 2007; Schmidt-Hieber et al., 2004). Days after newborn neuron birth, these cells respond to ambient GABA with tonic activation, due to the high concentration of intracellular chloride that leads to depolarization (Espo´sito et al., 2005; Ge et al., 2007). In 1–2 weeks, newborn neurons begin to receive synaptic GABAergic input. After 2–3 weeks, they begin to express glutamatergic receptors and, soon after, the direction of the chloride gradient switches such that GABAergic input results in hyperpolarization of newborn neurons (Espo´sito et al., 2005; Ge et al., 2007; Marı´n-Burgin et al., 2012). Around 1 month, new neurons receive synaptic glutamatergic Neuron 80, October 30, 2013 ª2013 Elsevier Inc. 593
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Perspective input from the entorhinal cortex, similar to mature cells (Deshpande et al., 2013; Li et al., 2012; Toni et al., 2007; Vivar et al., 2012). However, at this time point, new neurons have a lower density of GABA inputs and inhibitory postsynaptic currents (IPSCs) compared to those in mature granule neurons (Espo´sito et al., 2005; Li et al., 2012; Marı´n-Burgin et al., 2012). Once fully mature (about 8 weeks after birth), newborn neurons are essentially indistinguishable physiologically from developmentally born granule neurons. Because of these unique properties, young neurons are likely to be more excitable than mature neurons (Espo´sito et al., 2005; Mongiat et al., 2009; Mongiat and Schinder, 2011) and thus, in response to presynaptic inputs, the synapses formed by newborn neurons in the multisynapse boutons may be more dynamic than the existing synapses, contributing to the unique function of adult neurogenesis. There are still important aspects of this process that remain unknown, and a more complete understanding is critical to determining the influence of young neurons on the broader hippocampal circuit, as they are likely critical for both feedforward (to the CA3) and feedback (to the DG) inhibition. Functional Significance for Adult Neurogenesis Once the evidence for the existence of adult neurogenesis was generally accepted, the question of its functional relevance emerged. A series of correlational studies clearly revealed that increasing neurogenesis in the DG increased behavioral performance in a variety of hippocampus-related tasks and, conversely, decreasing neurogenesis resulted in behavioral impairments. Experiments designed to decrease neurogenesis by irradiation, viruses, antimitotic agents, or engineering transgenic animals whose adult neurogenesis could be regulated genetically or pharmacologically all confirmed a functional role for adult neurogenesis in the DG (Deng et al., 2010). To more completely understand the functional importance of adult neurogenesis, it is important to consider adult neurogenesis in the context of the hippocampus and its theoretical function as a whole. Individual GCs in the DG receive inputs from thousands of entorhinal cortex neurons, suggesting that they are capable of representing a highly complex combination of spatial and object features simultaneously. Several studies have suggested that the DG’s encoding role can be thought of in this fashion (Morris et al., 2013; O’Reilly and McClelland, 1994; Rolls and Kesner, 2006). Put into a computational perspective that has attracted considerable attention in recent years, the DG is critical for ‘‘pattern separation.’’ Pattern separation, as related to the DG, can be described as recoding cortical input information into a sparse, essentially orthogonal representation (McNaughton and Morris, 1987; Treves and Rolls, 1992). By manipulating the rate of adult neurogenesis, several groups of researchers have shown by ablating or overexpressing adult neurogenesis that newborn neurons are critical for making fine discriminations between neighboring spatial locations or highly similar environments in tests that reflect many of the computational characteristics of pattern separation (Clelland et al., 2009; Creer et al., 2010; Gu et al., 2012; Nakashiba et al., 2012; Sahay et al., 2011). 594 Neuron 80, October 30, 2013 ª2013 Elsevier Inc.
Together, these studies support the idea that a DG network dominated by young GCs is biased toward interpreting similar but not identical inputs as distinct, whereas older GCs are biased toward interpreting similar inputs as equivalent. While adult neurogenesis in the DG is now generally accepted to occur in all adult mammals, there are many mechanistic details about how it takes place that will need to be determined before we have a more complete understanding of its functional contribution to hippocampus-mediated behaviors. That said, we need to know a lot more about how hippocampal circuits mediate behaviors, and it is likely that understanding more about adult neurogenesis will contribute to a better understanding of hippocampal function. III. Reprogramming as a Path to Human NSCs and Neurons The field of stem cell biology changed forever when Takahashi and Yamanaka (Takahashi and Yamanaka, 2006) developed a simple and repeatable method to dedifferentiate mouse somatic cells (fibroblasts initially) to embryonic-like cells, termed induced pluripotent stem cells (iPSCs), that could give rise to every cell of the mouse body. The concept of reprogramming emerged from the early works of Briggs and King (Briggs and King, 1952) and Gurdon (Gurdon et al., 1958) but has become widely used as a technique since Takahashi and Yamanaka published their method and similar methods were shown to work for other species, including humans (Takahashi et al., 2007). A plethora of extensions and refinements followed, but the principle was established that essentially all cells in our body maintain an intrinsic plasticity for differentiating into a variety of cell types with completely different functions. The impact of this technology has been dramatic in all areas of biology but has been arguably most dramatic in the neurosciences. While much work remains to be done to improve and refine the technology, attempts to apply these techniques to the clinic are already ongoing. One could argue that it is too early to consider translational research because a much more basic understanding is required, but some of the applied approaches are inexorably pushing the field forward, resulting in the need for better systematic safety and reliability standards. Neurological and Psychiatric Disease Modeling Using Patient-Derived iPSCs Techniques using human embryonic stem cells (hESCs) have been available to researchers to develop methods for differentiating these cells to functional neurons of different classes or to overexpress mutant genes in the hESCs to model human disease (Marchetto et al., 2010b; Thomson et al., 1998). In addition, prior to the development of iPSC technology, somatic cell nuclear transfer (SCNT) was being applied in rare cases to study specific diseases (Rideout et al., 2002). However, soon after human cells were first reprogrammed (Takahashi et al., 2007), the modeling of neurodevelopmental and neurodegenerative diseases began in earnest, and the subsequent necessary effort to develop reliable protocols for differentiating the immature stem cells has progressed ever since. Neurogenetic disorders were modeled first (Dimos et al., 2008; Lee et al., 2009; Marchetto et al., 2010a; Zhang et al., 2010), followed by a few examples of sporadic and complex disorders (e.g., schizophrenia [SCHZ];
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Perspective Brennand et al., 2011; Paulsen et al., 2012; Pedrosa et al., 2011). While these modeling efforts are quite recent, concerns remain about the ability of reprogrammed fibroblasts to recapitulate disease phenotypes. Specifically, inadequate neuronal maturation, synaptic deficiency, and failed connectivity have been observed in many of the early-onset and neurodevelopmental diseases modeled so far (examples: familial dysautonomia [FD] [Lee et al., 2009], Rett syndrome [RTT] [Marchetto et al., 2010a; Ricciardi et al., 2012], Huntington’s disease [HD] [Chae et al., 2012], and SCHZ [Brennand et al., 2011]). It is possible that the apparent detection of synaptic deficits is partly the result of the types of measurements focused on so far. In neurodegenerative diseases and proteopathies, neuronal toxicity due to increased sensitivity to oxidative damage and proteasome inhibition seems to be more prevalent than strictly synaptic deficits. Examples include amyotrophic lateral sclerosis (ALS) (Mitne-Neto et al., 2011), Parkinson’s disease (PD) (Nguyen et al., 2011), Alzheimer’s disease (AD) (Israel et al., 2012), and Down syndrome, which mimics some aspects of AD (Shi et al., 2012). As the number of patients and types of neurological diseases being modeled increase, new patterns will emerge that could aid in developing earlier diagnostics tools and facilitate effective drug design. Significant interest among clinicians and the pharmaceutical industries has arisen as other neurological conditions are proposed to be modeled using iPSCs. Attractive candidate diseases include but are not restricted to bipolar disorder, major depression, multiple sclerosis, and idiopathic autism. Major Challenges in Modeling Neurological and Psychiatric Disease and Tools for Addressing Them When developing in vitro models, the main goal is to establish a meaningful parallel between the phenotypes observed in the dish and the disease pathology observed in vivo. An important set of challenges that currently surround this field involves the variability between clones and changes in clone genome and phenotype over passage and time. Targeted genome modification of hIPSCs using engineered constructs like zinc-finger nucleases (ZFNs) (Kim et al., 1996; Porteus, 2010), transcription activator-like effector nucleases (TALENs) (Bedell et al., 2012; Christian et al., 2010) and, more recently, clustered regularly interspaced palindromic repeats/CRISPR-associated (CRISPR/ Cas) system (Mali et al., 2013; Wiedenheft et al., 2012) present promising strategies for modeling monogenic and genetically defined disorders with reduced variability by generating isogenic control lines harboring defined genetic alterations (Soldner et al., 2011). For modeling sporadic diseases or complex neuropsychiatric disorders where there is no clear genetic etiology, the value of these targeted genomic approaches is less clear but still likely important. It is conceivable that identifying protocols that generate lineage-specific cells will solve this problem by allowing investigators to monitor the differentiation process more specifically. Defining and consistently obtaining the diseaserelevant neural cells at comparable levels of maturation should greatly reduce the phenotypic variability and highlight pertinent disease characteristics. Assessing neuronal network connectivity formation is important for understanding neuronal communication imbalance in disease but can be a challenging task because, as a general rule, the right subtype of neurons and the specific maturation time are not represented in the dish at
appropriate levels. To that end, designing cell-type-specific promoters may help in generating the desired populations of neurons that are directly involved in the disease being studied (for example, Hb9-positive cells for diseases involving alpha motor neurons such as ALS [Marchetto et al., 2008]). Additionally, single-cell expression profiling should further clarify the levels of population heterogeneity within in vitro cultures, and advances in media culture platforms and automated cell processing should provide the desired accuracy and consistency that will be required. For a number of neurological diseases, it remains unclear whether the phenotypes involved in the pathology are restricted to the neuronal population and to what extent the neighboring cells are also playing a major role. Improving the protocols for generation of cells present in the neuronal niche (i.e., astrocytes, oligodendrocytes, microglia, and endothelial cells) could reveal important disease phenotypes and contribute to the development of alternative therapies. Refining the techniques to analyze neuronal phenotypes will also help to detect more subtle differences. The field is driving strong interdisciplinary collaboration, bringing together technological advancements from multiple areas like electrical and mechanical engineering with principles of neuroscience and stem cell biology. New engineering and automation techniques are being applied to these types of studies as both engineers and the biotech industry and Big Pharma begin to explore and exploit this technology. Finally, we posit that many of the challenges facing disease modeling arise from the overall strategy employed. Many of the current disease modeling studies search for differences in gene expression generally or for basic functions that can be measured in vitro, i.e., functions that have been hypothesized to be correlated causally in the disease. Often these studies are not hypothesis driven but rather depend on existing techniques and the availability of somatic cells from whatever patients are available to the researcher. Researchers are beginning to work more closely with the clinicians who attend to and treat the patients to better understand the diversity of each of the patient populations to be studied and to obtain more restricted populations of patients (e.g., discordant monozygotic twins, drug-responsive versus nonresponsive cohorts, and severity of the disease). These kinds of collaborations between bench and bedside may not only lead to more targeted hypotheses but may also assist in decreasing the variability reported for in vitro modeling. While engineering platforms allow the researcher precision and control over the cellular microenvironment, in vivo transplantation of stem cell-derived populations of human pluripotent stem cells (hPSCs) and neurons into animal models presents a useful way to study human development and to model disease. Grafting NPCs at appropriate developmental stages could potentially utilize the myriad biochemical and biophysical cues provided in the endogenous niches to generate mature and functional populations of the desired cells. An excellent example is the transplantation of hPSC-derived forebrain NPCs into the neonatal mouse brain to generate cortical neurons with specific axonal projections and dendritic patterns corresponding to the native cortical neuron population (Espuny-Camacho et al., 2013). In addition, transplantation of hPSC-derived medial Neuron 80, October 30, 2013 ª2013 Elsevier Inc. 595
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Perspective ganglionic eminence (MGE) progenitors into the rodent brain produced GABAergic interneurons with mature physiological properties along an intrinsic timeline that mimics the endogenous human neural development (Nicholas et al., 2013). This emerging sector of stem cell biology has brought basic cell and molecular biologists together with engineers, clinicians, and large and small biotech companies. The new model organism is the human, and while this is a new field with plenty of caveats and unknowns, it is likely to stay around for the foreseeable future (Lancaster et al., 2013). IV. Applications of NSC to Disease and Therapy Human NSC Transplantation in Clinical Trials The discovery of the existence of NSCs throughout life in animals and then in humans led to rapid recognition of the therapeutic potential of these cells. Fetal human NSCs are an abundant source of neurons, astrocytes, and oligodendrocytes, with potential application to a variety of neurological conditions. Companies that were formed around the concept of transplanting human NSCs began the groundbreaking work of making clinical trials possible. Stem Cells, Inc. paved the way, generating clinical-grade banks of purified, fetal-derived human NSCs that are currently in use in clinical trials. They are being tested in patients with Pelizaeus-Merzbacher disease, a demyelinating condition of children that results in neurological dysfunction and death; the 2-year follow-up report indicates safety and improved and long-term myelination. These cells are also being tested in phase I/II clinical trials for spinal cord injury and the retinal disease dry age-related macular degeneration (AMD). In the latter case, human NSCs are not contemplated to replace the retinal pigment epithelial (RPE) cells that degenerate in AMD, as they do not generate that specific lineage but rather to substitute key RPE functions such as cytokine production and phagocytosis. Others pursuing the clinical application of human fetal NSCs include NeuralStem and the Azienda Ospedaliera Santa Maria, Terni, Italy. Both organizations are pioneering human fetal NSC transplants for ALS patients. Although it is early days, results thus far using well-defined human NSC products indicate that they can be transplanted safely and will integrate and generate long-lived progeny in their host. In contrast, the shocking report of tumor formation seen in a young Ataxia Telangiectasia patient given multiple mixed fetal human CNS grafts (Amariglio et al., 2009) cautions against the use of these cells outside of a clinical trial; furthermore, the disease indication should be carefully considered and tested in appropriate animal models to provide preliminary proof of concept and safety data before moving into humans. Given the rapid progress in pluripotent stem cell production of different neural lineages, one might ask whether fetal human NSC transplantation will at some point be superseded. The answer will depend on the relative safety profile of these different cell products and their ability to integrate and mature appropriately to provide efficacy. The next two decades will be revealing in this regard, and progress will be eagerly watched by patients and families afflicted by neurological disorders that could benefit from such transplants. Emphasis should be on performing well-designed clinical trials, and the NSC field must help educate patients to reduce the trafficking of unproven therapies. 596 Neuron 80, October 30, 2013 ª2013 Elsevier Inc.
PharmaNutri-Neurogenesis? NSCs exist throughout life in the hippocampal DG, but human VZ-SVZ stem cells stop actively generating neurons at about 2 years of age (Sanai et al., 2011). Adult hippocampal NSCs have life-long activity but their numbers decline in aging and are dramatically reduced in AD (Haughey et al., 2002), contributing to learning and memory deficits. Accumulated evidence that adult NSCs are responsive to environmental stimuli suggests ways to protect the activity of these cells, changing behavior and nutrition to enhance life-long NSC health. Psychiatric drugs can impact adult neurogenesis (Malberg et al., 2000), and small molecules could be designed more specifically for this target cell. Screening drugs for an impact on NSC function can help reduce toxicity and negative effects, such as the ‘‘chemobrain’’ side effects of some anticancer drugs (ElBeltagy et al., 2010). Growth factors that maintain NSC function are being explored as supplements, including infusion of IGF1, FGF, growth hormone, melatonin, and the BMP inhibitor Noggin (Bonaguidi et al., 2008), and the dramatic revitalization of aged hippocampal function demonstrated, for example, by loss of the Wnt inhibitor DKK1 (Seib et al., 2013) suggests that this will be a promising area for future translation. Utilizing Neural Progenitor Heterogeneity One particularly impressive example of the benefit of understanding progenitor heterogeneity is the progress made in treating childhood brain cancers. Comparison of in-depth gene expression analysis of normal murine progenitor cells and gene expression analyses of pediatric brain tumors has enabled the subdivision of medulloblastoma and ependymoma into different classes (Gibson et al., 2010; Johnson et al., 2010), providing more targeted therapies with better outcomes. In the future, a more complete understanding of human CNS progenitor subclasses will help to identify the cells responsible for different facets of neurological diseases and to target cell subsets more precisely to either enhance or diminish a particular cell group. Clinical Applications of Progenitor Cell Migration The long migrations of NSC progeny in vivo demonstrate that CNS cells have an astonishing capability to move about the nervous system. Receptor cytokines such as CXCR4-SDF1 guide normal migration and can be activated after trauma, e.g., in ischemic conditions, drawing cells out of adult germinal zones toward injured sites (Robin et al., 2006). While normal SVZ cells do not survive after attraction to these ischemic locations, it is possible that utilizing such homing mechanisms will help target therapeutic cells. One ongoing clinical trial utilizes the ability of immortalized NPCs to home to tumor sites; the cells are engineered to secrete a product that is activated once the cells reach glioblastoma lesions (Aboody et al., 2013). The powerful migratory ability of ventral forebrain-derived GABAergic neurons is being explored in translational studies that aim to deliver these cells therapeutically, anticipating that they will migrate and incorporate to dampen hyperexcitable states, for example, in epilepsy and spinal neuropathic pain (Bra´z et al., 2012; Maisano et al., 2009). One of the most astonishing examples of human NSC progeny migration is illustrated by human glial restricted precursors. When implanted into the shiverer demyelination mouse model, these cells produced oligodendrocytes that spread
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Perspective throughout the nervous system and essentially replaced the murine with human myelin (Windrem et al., 2008). Harnessing the mechanisms of neural migration will be valuable for more precise cell targeting and also to inhibit brain tumor-initiating stem cell spread. Enter Pluripotent Stem Cell-Derived NSCs Over the past 10 years, researchers have shown that the morphogenic gradients conceptualized and demonstrated in animal neurodevelopment are evolutionarily maintained and applicable to drive differentiation of diverse neural progeny from hPSCs. Stellar examples are the production of spinal motor neurons (Dimos et al., 2008; Wichterle et al., 2002) and midbrain substantia nigra dopaminergic (dA) neurons (Kriks et al., 2011). The story behind production of the latter cells underscores how critical it is to know the normal ontogeny. Dopaminergic cells were first derived from hPSCs by producing forebrain progenitors and then repatterning them to midbrain, but the resulting neurons largely lacked key markers of the midbrain A9 location desired for PD applications and were found to be unstable after transplantation into animal models. Instead, reiteration of the normal developmental track discovered in mouse, by first producing midbrain floor plate cells using high concentrations of SHH and then differentiating these into neurons, produced significantly more A9 dA neurons that were then shown to be stable and efficacious in animal models, providing the key proof of concept to move toward a clinical trial (Fasano et al., 2010; Kriks et al., 2011). While protocols for producing a diverse array of neural cells, both central and peripheral, are being developed, production of retinal cells has advanced, and subretinal transplantation of hESC-derived RPE cells for blindness disorders such as AMD is already in early phase clinical trials. The ability to produce a wealth of cell types from hPSCs for cell replacement and other potential therapies such as growth factors or drug delivery is undoubtedly exciting. One outstanding issue that has already been discussed, but worth noting again in this context, is that we still need to understand how to mature human NSC products appropriately. Our current approaches, which recapitulate early developmental morphogenic stimuli, are excellent at making early-stage cells, but many of the cell products remain immature, indicating that signals that normally extend development are lacking. Defining the stages and processes along the maturation arc is important, as these impact the ability of cells to integrate, migrate, and properly adopt desired characteristics, as seen for oligodendrocyte and photoreceptor transplantation (MacLaren et al., 2006; Warrington et al., 1993). If pluripotent cell-derived products are proven stable, safe, and efficacious when implanted into the human CNS, we can envision increasingly complex transplants, including not just RPEs but also photoreceptors, organoid-derived neural structures, or even 3D, tissue-printed personalized tissue grafts. The combination of engineering and stem cell research is powerful. Neural tissues that can self-assemble or be engineered from stem cells (Aoki et al., 2009; Eiraku et al., 2011; Kawamorita et al., 2002; Lancaster et al., 2013; Meyer et al., 2011) have a promising future to model complex, multicell neural diseases and as a basis for toxicity testing and mechanistic studies. The enormous advantage of having human neural cells widely avail-
able is something we could only dream about 25 years ago, and we predict that they will prove even more valuable and will likely supplant animal testing in efficacy studies, which have failed to model many human diseases; however, there is much work ahead to achieve this worthwhile goal. Given the rapid upward trajectory of biotechnological and biomedical advances, we can afford to let our imaginations range: will biological devices that incorporate cells and materials be developed, for example, as retinal prostheses or treatments for epilepsy or PD? Will we be protected by bioengineered sensors that use neural and computer elements, a ‘‘canary on a chip,’’ to detect stroke or external toxins? The future for the next generation of NSC researchers and for NSC translation is bright. Conclusion Thanks to great strides in our ability to observe and study germinal cells, and to investigate how neurons and glia are generated at cellular and molecular levels, we now have an impressive body of knowledge concerning NSC biology. Many of the foundational problems concerning NSCs were soluble only after a specific tool was developed (Table 1) and, with the extraordinary blossoming of technologies that is currently ongoing (Table 2), much more information is anticipated concerning the wealth of NSC types and their regulation. As imaging technologies advance, we should make significant headway in understanding how NSCs behave within endogenous niches and after implantation in vivo. Animal studies, notably in mouse, will continue to provide pioneering advances, especially to test application of new tools, but increasingly, we see the field moving toward pursuing the study of human NSC biology. The astonishing success of reprogramming somatic cells into neuronal and glial progeny with just a handful of genes (Najm et al., 2013; Vierbuchen et al., 2010) has made almost any cellular change seem possible, and the more we know about how NSCs tick, the better chance we have to produce, on demand, bona fide human neurons and glia for a multitude of in vivo and ex vivo applications. Overall, it has been inspiring to witness the extraordinary growth of knowledge in this area and to contribute to what is now an established field of NSC research, with great potential for advancing our understanding and healing of that most intricate organ, the nervous system. ACKNOWLEDGMENTS We would like to thank Qingjie Wang, Mary Lynn Gage, and Carol Marchetto for help in preparing the manuscript. F.H.G. is a founder for Stem Cells, Inc. and a member of the Scientific Advisory Board. S.T. is a founder of StemCulture, Inc. REFERENCES Aboody, K.S., Najbauer, J., Metz, M.Z., D’Apuzzo, M., Gutova, M., Annala, A.J., Synold, T.W., Couture, L.A., Blanchard, S., Moats, R.A., et al. (2013). Neural stem cell-mediated enzyme/prodrug therapy for glioma: preclinical studies. Sci. Transl. Med. 5, 84ra59. Aimone, J.B., Wiles, J., and Gage, F.H. (2009). Computational influence of adult neurogenesis on memory encoding. Neuron 61, 187–202. Altman, J. (1962). Are new neurons formed in the brains of adult mammals? Science 135, 1127–1128.
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Perspective Neural Stem Cells: Generating and Regenerating the Brain Fred H. Gage1,* and Sally Temple2 1Laboratory
of Genetics, The Salk Institute for Biological Studies, 10010 N. Torrey Pines Road, La Jolla, CA 92037, USA Stem Cell Institute, One Discovery Drive, Rensselaer, NY 12144, USA *Correspondence: [email protected] http://dx.doi.org/10.1016/j.neuron.2013.10.037 2Neural
One of the landmark events of the past 25 years in neuroscience research was the establishment of neural stem cells (NSCs) as a life-long source of neurons and glia, a concept that shattered the dogma that the nervous system lacked regenerative power. Stem cells afford the plasticity to generate, repair, and change nervous system function. Combined with reprogramming technology, human somatic cell-derived NSCs and their progeny can model neurological diseases with improved accuracy. As technology advances, we anticipate further important discoveries and novel therapies based on the knowledge and application of these powerful cells. Introduction It is an understatement to say that the conceptual advances and practical applications of stem cell research have been game changers for the field of neuroscience. When the term ‘‘stem cell’’ was used 25 years ago, the context was typically adult tissue homeostasis, mouse genetics, or early development. Thanks to several landmark advances in the area of developmental neurobiology, neural stem cells (NSCs) are now central to our discussion of how the brain forms. While there was evidence for adult neurogenesis early on, it was controversial, and the underlying cells responsible for continued neuronal and glial production were not characterized. Today it would be difficult to consider adult neurogenesis without reference to endogenous NSCs and their niches. Although early researchers had determined that individual transcription factors directed cell fate, as in MyoD for muscle, and had done pioneering experiments proving that oocyte proteins could dedifferentiate a somatic cell nucleus, they could not have imagined the explosion of reprogramming that now allows us to generate human neural cells from induced stem cells and enables us to model nervous system diseases in entirely new ways. Progress at the basic research level has also been astonishing, and we are already witnessing the translation of NSC science, with several clinical trials ongoing and more in the planning stages. In the following Perspective, we will review some of the milestones of the last 25 years in NSC research. Rather than providing a comprehensive review of these advances, we intend to highlight the major events and discoveries that we feel have made the most important contributions to our field. In particular, we will focus on the shifts in the field around the concept of adult neurogenesis and stem cells in the adult brain, especially in the hippocampus. We will discuss the more recent development of methodologies for reprogramming and induced pluripotent stem cells (iPSCs) and outline our views on the promise of NSC-based approaches for the treatment of disease. Significant milestone advances that have driven NSC research forward have been summarized in Table 1, and we have also provided a tools wish list that would enable researchers to address some key 588 Neuron 80, October 30, 2013 ª2013 Elsevier Inc.
remaining questions concerning NSC biology (Table 2). The views here represent our personal perspectives on what has been particularly significant; we readily acknowledge that this only reflects a fraction of the interesting and important work in the field, and we apologize to those whose work we have not had space to discuss and reference. As you read this Perspective, we hope to inspire you to imagine the conceptual advances and new applications of NSC research over the next 25 years. I. Early Halcyon Days for Studies on the Development of the Nervous System It is difficult to imagine how much in the dark we were about mammalian nervous system development back in the 1980s. One of the burning questions at that time was whether or not common progenitor cells for neurons and glia even existed. Stem cells were not generally considered a part of brain development but rather the building blocks of other, more plastic tissues. The tools available to us to address these fundamental questions were limited. In vivo studies of the developing brain relied heavily on histological stains that would have been familiar to Camillo Golgi, in vitro culture media were primitive, growth factors to stimulate division of neural progenitors had not been identified, and there were only a handful of antibodies to identify neural cell types. But we were young researchers in a young field, excited by the questions, undaunted by the challenges, and fortunate to be on hand at the beginning of arguably the most extraordinarily productive quarter century in developmental neuroscience. Identifying the Progeny of Single Cells Revealed Heterogeneous Progenitor Cells, Including NSCs As is often the case in developmental studies, invertebrate research had taken the lead, and methods to genetically mark individual progenitor cells in vivo revealed unexpected clonal boundaries and patterns that revolutionized our comprehension of how insects are built. With this inspiration, the question was how to look into the developing CNS of large, cytologically complex, opaque organisms that inconveniently developed in utero. The door was opened by the development of retroviral
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Perspective lineage-tracing methods applicable to mammals. In the mouse cerebral cortex, for example, these techniques showed that most progenitor cells at midgestation generated surprisingly small clones, typically solely of neurons, that could migrate quite widely (Price and Thurlow, 1988; Turner and Cepko, 1987; Walsh and Cepko, 1992). Earlier infected clones could span multiple cortical layers, indicating multipotency for neuronal production (Kornack and Rakic, 1995). We learned that mixed neuron-glia clones were rare in the cortex but far more numerous in the spinal cord, where clones containing motor neurons, interneurons, and glial progeny were observed (Leber and Sanes, 1995). To determine whether the diversity of clones seen in vivo reflected environmental cues that the clonal progeny encountered or cell-intrinsic heterogeneity, researchers began to perform clonal studies in vitro, observing single embryonic cells growing in a standardized environment to uncover cell-based differences. Most embryonic progenitors produced clones of solely neurons, others of solely glia, but, most excitingly, a small subset of progenitors had large proliferative potential and incontrovertibly produced both neuronal and glial progeny. These progenitors also demonstrated some capacity for self-renewal, a cardinal property of stem cells (Anderson, 1989; Cattaneo and McKay, 1990; Davis and Temple, 1994; Kilpatrick and Bartlett, 1993; Temple, 1989). These discoveries led to the concept that nervous system development, both peripheral and central, was similar to that of the blood system and relied on fundamental, multipotent NSCs that repeatedly produced more restricted neural progenitors that, in turn, divided just a few times to produce small numbers of differentiated progeny. Today, while studies of progenitor heterogeneity are advancing—for example, by the identification, within germinal zones, of cells with diverse morphologies and diverse markers (Ayoub et al., 2011; Fietz et al., 2012; Franco et al., 2012; Kawaguchi et al., 2008; Pinto et al., 2008)—embryonic neural progenitor cells (NPCs) are still classified into just two basic types: multipotent NSCs and transit amplifying/intermediate progenitor cells (IPCs) (Englund et al., 2005). We have yet to define most of the progenitor subsets contributing the vast array of fates seen clonally in vivo and in vitro and to understand their key regulators and their role in neural development. These will be significant goals for the next decade. Uncovering the Dynamics of NSC Behavior Development can be thought of as increasing cellular complexity over time. We marvel at the precise orchestration of cell proliferation and then differentiation into innumerable types of neurons and then glia. How are these events choreographed? Pioneering heterochronic transplantation studies demonstrated that early progenitors have a wide multipotency but late progenitors are unable to produce the earlier fates (Frantz and McConnell, 1996; McConnell and Kaznowski, 1991). This finding led to a key idea that the potential of CNS stem cells is progressively, temporally restricted. How do CNS progenitor cells change over time? The development of tools to record extended time-lapse movies of CNS germinal cells ex vivo has yielded enormous insights. Movies of isolated cortical clones growing in 2D cultures showed that the lineage trees of isolated murine CNS progenitor cells were highly reminiscent of those of invertebrates and, astonishingly,
that individual cells were programmed to recapitulate the timing of diverse progeny seen in vivo, including their gradual restriction in potency (Shen et al., 2006). Combining retroviral labeling and slice culture, we could observe cortical progenitor cells in a system that retained much of the normal 3D niche architecture. This technique revealed that radial glial cells (RGCs), which span from their soma in the ventricular zone (VZ) to the pial surface, were the fundamental progenitor cells for neurons (Noctor et al., 2001) and later glia. Combined with in vitro studies using transgenic reporters for RGCs (Malatesta et al., 2000), this finding led to the notion that embryonic multipotent CNS NSCs were a subset of RGCs. The advancement of sophisticated imaging techniques and analytical tools (Winter et al., 2011) has great potential to further illuminate progenitor cell behavior over time. We look forward to observing multiple signals simultaneously, enabling us to follow the expression and movements of not only single genes or proteins but also of pathways and networks, as the progenitor cells change during neural development and after challenges. Much progress has been made to understand the temporal control of NSC output. Steps in the timing process rely on production of gliogenic cues, such as cardiotropin, transcription factor sequences, DNA methylation changes, and chromatin modifications (Barnabe´-Heider et al., 2005; Hirabayashi et al., 2009; Namihira et al., 2009; Pereira et al., 2010). Yet today, key aspects of the mechanisms that underlie progenitor temporal control remain enigmatic, presenting a challenge that is somewhat ahead of the tools currently available. We need the means to rapidly record dynamic changes in multigene expression, transcription factor binding, and chromatin structure and, ideally, to do so within living progenitor cells as they divide and move down the lineage trees. It is a lot to ask but, given the rapid evolution of single-cell tools, we might get there sooner than expected. How Are CNS Stem Cells Regionally Patterned? One of the central discoveries in developmental neuroscience that has emerged in this past 25-year era concerns how the nervous system is regionally patterned. Embryological manipulations—first in chick, then with transgenic mice—elucidated the morphogenic gradients that pattern neural tissue, for example, ensuring that motor neurons and oligodendrocytes arise ventrally and interneurons arise dorsally in the spinal cord (Briscoe et al., 1999; Liem et al., 1997). Other notable studies revealed that specific CNS regions can be organizers; for example, the midhindbrain isthmus drives midbrain patterning via release of FGF8, so that implanted beads containing FGF8 cause duplication of the cerebellum (Martinez et al., 1999). Studies of mouse mutants that were almost perfect apart from the lack of specific brain regions showed that the CNS develops as modules defined by transcription factor domains (Puelles and Rubenstein, 2003). One fascinating question that we have yet to answer is how morphogenic gradients intersect with and activate specific lineage programs in NSCs and their progeny, so that discrete, regionally appropriate progeny are made. While CNS development is modular, cells can cross regional boundaries. In a landmark demonstration, GABAergic neurons in the forebrain were shown to be born ventrally and migrate into the overlying dorsal cortex (Anderson et al., 1997). This Neuron 80, October 30, 2013 ª2013 Elsevier Inc. 589
Proliferation markers: tritiated cell labeling, Brdu, Edu, Ki67
Neural cell-type-specific antibodies
In situ hybridization of mRNAs in CNS germinal zones
Viral vectors for lineage tracing and gene manipulation
Single-cell cloning for CNS neuronal and glial progenitor cells
Confocal microscopy
Surface marker definition and cell separation
Serum-free culture methods with identified NSC growth factors
Long-term time-lapse microscopy
Conditional mutations in mice
Microarrays
RNA interference (microRNA, siRNA, shRNA)
hESCs SCNT
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1970s FACS; 1989 MACS
1990s
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Tools
1961
Approximate Impact Start Date
590 Neuron 80, October 30, 2013 ª2013 Elsevier Inc. How are neural progeny derived from human pluripotent cells?
How does gene knockdown in vitro and in vivo alter NSC behavior?
What genes are expressed in NSCs?
What is the impact of gene LOF or GOF on NSCs?
What is the identity of neuronal and glial progenitor cells in 3D slices?
Which factors stimulate NSC proliferation and differentiation in vivo?
What are the major subtypes of embryonic and adult progenitor cells?
RNAi library screening to identify NSC regulators
Gene expression after perturbations?
How are NSCs regionally patterned?
What changes occur during CNS germinal cell division?
Which factors expand progenitor types ex vivo?
How do prospectively isolated NSCs/NPCs behave?
3D tissue imaging
What is the relationship of NSC subtypes to brain tumor-initiating cells?
What are the lineage trees of individual NSCs and NPCs?
How do environmental factors impact NPC/NSC behavior?
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Production of RPE cells and other neural products for clinical trials
RNA interference therapeutics
Production of diverse neural progeny from NSCs and PSCs
Exogenous factors to enhance NSC activity
Enrichment of progenitor cells for transplantation
Pathology
Do self-renewing NSCs exist and in which CNS region?
What intrinsically different types of NSCs and NPCs exist? Identity of subcellular NSC markers in vivo
Targeted gene therapy
How do progenitors generate progeny in slice culture?
What progeny do single progenitors generate in vivo? Are NSCs functional?
Are there common progenitor cells for neurons and glia?
Pathology
How do NSCs respond to disease, drugs, and environmental perturbations?
Antibodies to select cells for transplant; drugs that impact stem cell growth, e.g., anti-angiogenic Abs
Assessment of cell growth after cell transplantation
Translation
What genes are expressed by NSCs, NPCs, and their progeny?
How do NSCs respond to disease, drugs, and environmental perturbations?
How is NSC proliferation regulated by environment?
What types of progeny do NSCs produce?
Do NSCs exist in the adult?
How are germinal niches structured in vivo?
What is the birthdate of neural cells during development?
Questions
Table 1. Milestones in NSC Research Related to Technology Development and Application
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Perspective
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Gene repair; creation of appropriately matched control and disease lines
How is chromatin modified in NSCs and their progeny?
Can somatic cells be turned into cells resembling embryonic stem cells?
What is the RNA complement of NSCs and their progeny?
Can somatic cells be converted directly into neurons and NSCs?
Does specific gene alteration impact NSC behavior?
Chip-seq Gro-seq
Mouse, human iPSCs
RNA-seq Gro-seq
Direct reprogramming
TALEN and CRISPR gene editing
2005
2006–2007
2008
2010
2010–2013
What genes are critical to specify neural fates?
What is the functional effect of optogenetic engineering in NSCs? Optogenetics 2002
Transcriptome studies after perturbations
Questions Tools Approximate Impact Start Date
Table 1.
Continued
How do chromatin alterations alter NSC behavior?
Report gene expression more accurately
Rapid generation of purer cell populations
Disease-related changes in RNA expression
Neural disease modeling; drug screening; potential autologous transplantation
Diagnosis of chromatin changes in disease; development of drugs to target
To stimulate NSC progeny after transplantation
Translation
Perspective finding—that almost the entire inhibitory neuron complement of the cortex arose from NSCs that were born elsewhere—was most surprising. Migration was not just along radial glia but tangential (O’Rourke et al., 1995), and the routes of all sorts of peripatetic CNS progenitor cells have now been revealed, from the pioneering Cajal-Retsius neurons from the cortical hem (Bielle et al., 2005) to the vast spreading migrations of different waves of oligodendrocyte precursors (Kessaris et al., 2006; Timsit et al., 1995). Such mixing increases the richness of connective possibilities, and cell migratory defects will continue to be explored as the cause of multiple neurological disorders. Building the Foundation of Human NSC Studies Much of our understanding of mammalian neural development comes from mouse studies, and resources such as BGEM, Genepaint, the Allen Brain Atlas, MGI, and KOMP enable us to question further and deeper. Still, the mouse is lissencephalic, its neuronal complement is born in essentially 7 days, and no one doubts comparative studies that indicate significant differences in how the 1,000-fold larger human brain is built over 9 months of gestation (Zeng et al., 2012). But, thanks to vast improvements in tools and resources and to the advent of human stem cell biology, we are now ready to take on the task of understanding human CNS development. The next 25 years, we predict, will witness great strides in that area. Already, we know that the human forebrain has not just the VZ and subventricular zone (SVZ) but also a significantly expanded germinal zone, the outer SVZ, which helps account for the orders of magnitude increase in its size and complexity (Bystron et al., 2008; Hansen et al., 2010). Dissection of these germinal layers provides a clue to the key transcription factors and pathways that characterize their constituent cells (Fietz et al., 2012). In the future, fundamental molecular studies will expand our knowledge of the temporal patterns of gene expression and epigenomic changes that accompany human neural development (Kang et al., 2011). New techniques for creating in vitro human neural organoids with salient morphologic features such as retinal and cortical layering (Aoki et al., 2009; Eiraku et al., 2011; Lancaster et al., 2013; Meyer et al., 2011) will enable 3D imaging of how human CNS progenitor cells work and will broaden our understanding of CNS morphogenesis. Progress over the past 25 years in characterizing embryonic NSCs and understanding their patterning, lineages, and role in nervous system development has been and continues to be complemented by tremendous strides in the characterization of adult NSCs, enabling cross-fertilization of ideas and tools and encompassing adult learning and memory, environmental regulation, cancer, and aging. II. Discovery and Elaboration of Adult NSCs and Neurogenesis History: Cell Division to Neurogenesis in the Adult Brain The observations of cell division and differentiation in the adult brain emerged from studies of brain development and were greatly advanced by the early application by Leblond and colleagues of tritiated thymidine, which incorporates into the DNA of dividing cells and can be detected by autoradiography. Using this labeling technique, Leblond and colleagues observed and concluded that glial cells were probably dividing throughout Neuron 80, October 30, 2013 ª2013 Elsevier Inc. 591
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Perspective Table 2. Wish List of Technologies that Will Accelerate or Enable Important Directions in NSC Research Tool
Questions
Translation
More antibody and other markers to prospectively identify NSCs and NPCs
What is the range of types of NSCs and NPC present in mouse and human?
Understanding the contribution of NSC lineages to disease
Diverse fluorescent markers to report multiple genes, multiple proteins
Visualize complex elements in the same cell
Broad multiplex imaging: for immunostaining, in situ and gene expression reporting
To track multiple molecules simultaneously—analysis of networks, pathways and multimolecular complexes
How do NSC pathways change during development and with aging?
3D cultures that recapitulate human neural development; integrate engineering
What mechanisms underlie human NSC development into neural progeny?
What morphologic changes accompany CNS development?
Improved cell transplants and prostheses and disease models
More 2D and 3D imaging and analysis tools ex vivo
How do NSCs interact with niche cells, statically and dynamically?
Quantification of image information
Imaging standards for cell products; pathology assessment
High definition (deeper and higher resolution), in vivo imaging
What is the dynamic behavior of NSCs in vivo in the normal niche?
How do NSCs migrate and integrate in vivo?
Precise cell tracking after transplantation in vivo
Closer collaborations between basic, clinical, applied, and commercial stake holders
What are the clinical implications of NSC-based discoveries?
Critical for effective translation
Better nonviral transfection methods
Accelerate and reduce the cost of answering many questions about NSCs
Allow easy, high-throughput screening
Improved models of neurological disease
What is the disease mechanism?
the parenchyma (Smart and Leblond, 1961). They specifically found dividing cells in the subependymal zone (SEZ) but did not observe neurogenesis because the percursors born in the SEZ, later renamed the SVZ, must migrate to the olfactory bulb before they differentiate into neurons. Soon after these pioneering studies, Joe Altman, using the same techniques, observed dividing cells in the subgranular zone and speculated that neurogenesis occurred in the adult rat and cat dentate gyrus (DG) (Altman, 1962, 1963). Then, in 1965, he and Gopal Das provided the first strong evidence for neurogenesis in the adult brain (Altman and Das, 1965), reporting on the migration of cells that were born postnatally in the SVZ and matured into neurons in the olfactory bulb. In 1969, Altman was the first to describe the rostral migratory stream, located between the SVZ and olfactory bulb (Altman, 1969). Surprisingly, there was little follow-up to these discoveries for almost 10 years, when Kaplan and Hinds provided electron microscopic data, combined with tritiated thymidine staining, that showed the existence of adult-born neurons in the DG and the olfactory bulb (Kaplan and Hinds, 1977). The next phase in the early history of adult neurogenesis moved to the avian brain, where Goldman and Nottebohm first detected what they reported was neurogenesis in adult birds (Goldman and Nottebohm, 1983); Paton and Nottebohm then demonstrated functionality by unit recording and then autoradiography of thymidine-labeled neurons (Paton and Nottebohm, 1984). After another period of little activity in the area, four developments and discoveries changed the perception of neurogenesis in the mammalian brain in the 1990s. The first was the observation that proliferation levels of the early progenitor cells and subsequent numbers of newborn neurons were regulated. Gould, 592 Neuron 80, October 30, 2013 ª2013 Elsevier Inc.
What pathways are best targeted?
Could supplant animal models for efficacy and some tox studies
Cameron, and McEwen demonstrated that stress levels negatively affected the numbers of proliferating cells in the DG (Gould et al., 1992). This finding was followed by a series of observations demonstrating that neurogenesis could be substantially increased by running (van Praag et al., 1999), that housing animals even for short periods of enrichment in complex environments increased robustly the number of surviving newborn neurons (Kempermann et al., 1997), that learning itself could influence adult neurogenesis (Do¨bro¨ssy et al., 2003; Gould et al., 1997), and that antidepressant drugs (SSRIs) as well as alcohol (Nixon and Crews, 2002) could influence components of the adult neurogenesis process (Malberg et al., 2000). Around this same time, neurogenesis was shown to decrease with age but persist throughout life (Kuhn et al., 1996). A second development was the advancements in immunohistological techniques, combined with the application of confocal microscopy to the study of adult neurogenesis and, importantly, the application of stereological techniques for labeling dividing cells (in particular bromodeoxyuridene [BrdU]) and neurons (initially NeuN). These techniques allowed the simultaneous colabeling of neurons and proliferating cells and quantification of the changes in these cells in vivo, convincingly demonstrating that the dividing cells in the DG indeed became neurons (Kempermann et al., 1997; Kuhn et al., 1996, 1997). Using these techniques combined with transplantation, Lois and AlvarezBuylla demonstrated that endogenous and engrafted SVZ cells migrated into the olfactory bulb (Lois and Alvarez-Buylla, 1994). They also provided evidence for the surprising finding that stem cells in the adult SVZ expressed the astrocyte marker GFAP (Doetsch et al., 1999).
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Perspective The third important advance was the application of these newly applied techniques to identify new neurons in the DG of cancer patients who were given BrdU for diagnostic purposes (Eriksson et al., 1998), generalizing the findings of adult neurogenesis to humans. This demonstration of human adult neurogenesis was confirmed recently using carbon14 dating techniques on neurons derived from individuals born during the period of above-ground nuclear testing (Spalding et al., 2013). The fourth important development was the identification of methods to isolate, propagate, and differentiate progenitors from the adult CNS in defined culture conditions. This breakthrough was first achieved from dissections of the lateral wall of the striatum to obtain cells of the SVZ and then the expansion of the proliferating population into what came to be known as neurospheres (Kilpatrick and Bartlett, 1993; Reynolds and Weiss, 1992). The subgranular zone (SGZ) population of dividing cells was isolated from the hippocampus and then expanded in vitro and maintained as monolayers (Palmer et al., 1995, 1997). The ability to isolate, maintain, expand, and differentiate these precursor cells in vitro led to the ability to explore, in more detail, the cellular and molecular nature of the cells and the mechanisms that regulated their behavior. The in vitro cells could then be tested in vivo using the newly developed in vivo tools. The demonstration of neurogenesis in humans, along with its regulation by behavior and the environment, highlighted its relevance to the scientific community and helped motivate research into the wider regenerative potential of NSCs. The Niche Over the ensuing decade (2000–2010), many of the details of the phenomenon of neurogenesis were revealed. Importantly, the anatomical location and cellular constituents of the ‘‘niche’’ where NSCs are born and maintained were found to be more complex than anticipated but to be similar to niches that were being discovered for stem cells generated in other adult organs. The phenomenon of neurogenesis can be delineated into four processes: cell proliferation, migration, cell survival, and neuronal differentiation. Each aspect is critical to the overall levels of neurogenesis. For example, NPC proliferation occurs in other regions of the adult brain but NPCs do not differentiate into neurons there, either maintaining the properties of precursors or becoming glia. However, NPCs isolated from these nonneurogenic regions, such as cortex and optic nerve, in the adult brain retain the potential to become neurons in vitro when expanded in FGF-2 and treated with differentiating molecules like retinoic acid and Forskolin, indicating that extrinsic factors play a major role in stimulating NPCs to differentiate into neurons (Palmer et al., 1999). Additional support for the importance of the neurogenic microenvironment comes from the finding that NPCs located in the SVZ and SGZ are the only ones that adopt a neuronal cell fate under normal physiological conditions in the adult brain; however, if these NPCs are isolated from the SVZ or GVZ with the techniques described above and then transplanted into ectopic regions of the adult brain, they differentiate mostly to oligodendrocytes and astrocytes (Seidenfaden et al., 2006). In contrast, NPCs from the DG can differentiate into olfactory bulb neurons when grafted to the SVZ (Suhonen et al., 1996) and, more dramatically, NPCs isolated from a nonneurogenic re-
gion, such as the spinal cord, can differentiate into neurons when transplanted into the DG, supporting the idea that external cues from the local microenvironment promote the neuronal differentiation of NPCs (Shihabuddin et al., 2000). The SVZ and SGZ represent neurogenic niches or local microenvironments that permit and support neurogenesis. To date, many of the cellular constituents of the niche have been identified, including astrocytes (Song et al., 2002b), endothelial cells (Shen et al., 2004), microglia (Sierra et al., 2010), and the blood vascular system itself (Palmer et al., 2000). A more complete understanding of the molecules and events that regulate the niche and its influence on neural stem cell behavior is being revealed on a daily basis in the current literature. What will be very useful—and has not yet been achieved because of the optical limits—is the observation in real time of stem cells in their niche and the temporal process by which the cells interact with their microenvironment to generate neurons. Morphological Transition of Stem Cells to Neurons New neurons born in the adult brain undergo a maturation process that takes several months before they are essentially equivalent to mature neurons. Arising from a local stem cell population (Gage, 2000), adult-born neurons initially are not directly connected at all to local circuitry. Nonetheless, they are apparently responsive to local neurotransmitters, likely through spillover from nearby synapses (Song et al., 2002a). It takes about 2 months for newborn neurons to reach morphological maturity. Although no significant structural differences are observed between fully mature adult-born and perinatal-born neurons, the maturation process is delayed in the adult (Zhao et al., 2006), and it is very likely that this delay is crucial for their function both as young neurons and subsequently as mature neurons (Aimone et al., 2009). Notably, the spine formation process of adult-born neurons appears to be different from that of perinatal-born neurons in that adult-born neurons preferentially target pre-existing synapses; little is known about the underlying mechanisms (Toni et al., 2007). One hypothesis is that glutamate spillover may play a chemoattractive role and induce filopodia growth toward active synapses (Toni and Sultan, 2011; Toni et al., 2007). Local synaptic activity may induce glutamate release and activate glutamate receptors in filopodia, which induces new filopodia to target the existing synapse (Toni and Sultan, 2011). This structural difference parallels the differences between the physiologies of immature and mature granule cells. Newborn neurons display a high input resistance (Espo´sito et al., 2005), receive less inhibition (Li et al., 2012), and have been shown to exhibit considerably greater synaptic plasticity than mature granular neurons (Ge et al., 2007; Schmidt-Hieber et al., 2004). Days after newborn neuron birth, these cells respond to ambient GABA with tonic activation, due to the high concentration of intracellular chloride that leads to depolarization (Espo´sito et al., 2005; Ge et al., 2007). In 1–2 weeks, newborn neurons begin to receive synaptic GABAergic input. After 2–3 weeks, they begin to express glutamatergic receptors and, soon after, the direction of the chloride gradient switches such that GABAergic input results in hyperpolarization of newborn neurons (Espo´sito et al., 2005; Ge et al., 2007; Marı´n-Burgin et al., 2012). Around 1 month, new neurons receive synaptic glutamatergic Neuron 80, October 30, 2013 ª2013 Elsevier Inc. 593
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Perspective input from the entorhinal cortex, similar to mature cells (Deshpande et al., 2013; Li et al., 2012; Toni et al., 2007; Vivar et al., 2012). However, at this time point, new neurons have a lower density of GABA inputs and inhibitory postsynaptic currents (IPSCs) compared to those in mature granule neurons (Espo´sito et al., 2005; Li et al., 2012; Marı´n-Burgin et al., 2012). Once fully mature (about 8 weeks after birth), newborn neurons are essentially indistinguishable physiologically from developmentally born granule neurons. Because of these unique properties, young neurons are likely to be more excitable than mature neurons (Espo´sito et al., 2005; Mongiat et al., 2009; Mongiat and Schinder, 2011) and thus, in response to presynaptic inputs, the synapses formed by newborn neurons in the multisynapse boutons may be more dynamic than the existing synapses, contributing to the unique function of adult neurogenesis. There are still important aspects of this process that remain unknown, and a more complete understanding is critical to determining the influence of young neurons on the broader hippocampal circuit, as they are likely critical for both feedforward (to the CA3) and feedback (to the DG) inhibition. Functional Significance for Adult Neurogenesis Once the evidence for the existence of adult neurogenesis was generally accepted, the question of its functional relevance emerged. A series of correlational studies clearly revealed that increasing neurogenesis in the DG increased behavioral performance in a variety of hippocampus-related tasks and, conversely, decreasing neurogenesis resulted in behavioral impairments. Experiments designed to decrease neurogenesis by irradiation, viruses, antimitotic agents, or engineering transgenic animals whose adult neurogenesis could be regulated genetically or pharmacologically all confirmed a functional role for adult neurogenesis in the DG (Deng et al., 2010). To more completely understand the functional importance of adult neurogenesis, it is important to consider adult neurogenesis in the context of the hippocampus and its theoretical function as a whole. Individual GCs in the DG receive inputs from thousands of entorhinal cortex neurons, suggesting that they are capable of representing a highly complex combination of spatial and object features simultaneously. Several studies have suggested that the DG’s encoding role can be thought of in this fashion (Morris et al., 2013; O’Reilly and McClelland, 1994; Rolls and Kesner, 2006). Put into a computational perspective that has attracted considerable attention in recent years, the DG is critical for ‘‘pattern separation.’’ Pattern separation, as related to the DG, can be described as recoding cortical input information into a sparse, essentially orthogonal representation (McNaughton and Morris, 1987; Treves and Rolls, 1992). By manipulating the rate of adult neurogenesis, several groups of researchers have shown by ablating or overexpressing adult neurogenesis that newborn neurons are critical for making fine discriminations between neighboring spatial locations or highly similar environments in tests that reflect many of the computational characteristics of pattern separation (Clelland et al., 2009; Creer et al., 2010; Gu et al., 2012; Nakashiba et al., 2012; Sahay et al., 2011). 594 Neuron 80, October 30, 2013 ª2013 Elsevier Inc.
Together, these studies support the idea that a DG network dominated by young GCs is biased toward interpreting similar but not identical inputs as distinct, whereas older GCs are biased toward interpreting similar inputs as equivalent. While adult neurogenesis in the DG is now generally accepted to occur in all adult mammals, there are many mechanistic details about how it takes place that will need to be determined before we have a more complete understanding of its functional contribution to hippocampus-mediated behaviors. That said, we need to know a lot more about how hippocampal circuits mediate behaviors, and it is likely that understanding more about adult neurogenesis will contribute to a better understanding of hippocampal function. III. Reprogramming as a Path to Human NSCs and Neurons The field of stem cell biology changed forever when Takahashi and Yamanaka (Takahashi and Yamanaka, 2006) developed a simple and repeatable method to dedifferentiate mouse somatic cells (fibroblasts initially) to embryonic-like cells, termed induced pluripotent stem cells (iPSCs), that could give rise to every cell of the mouse body. The concept of reprogramming emerged from the early works of Briggs and King (Briggs and King, 1952) and Gurdon (Gurdon et al., 1958) but has become widely used as a technique since Takahashi and Yamanaka published their method and similar methods were shown to work for other species, including humans (Takahashi et al., 2007). A plethora of extensions and refinements followed, but the principle was established that essentially all cells in our body maintain an intrinsic plasticity for differentiating into a variety of cell types with completely different functions. The impact of this technology has been dramatic in all areas of biology but has been arguably most dramatic in the neurosciences. While much work remains to be done to improve and refine the technology, attempts to apply these techniques to the clinic are already ongoing. One could argue that it is too early to consider translational research because a much more basic understanding is required, but some of the applied approaches are inexorably pushing the field forward, resulting in the need for better systematic safety and reliability standards. Neurological and Psychiatric Disease Modeling Using Patient-Derived iPSCs Techniques using human embryonic stem cells (hESCs) have been available to researchers to develop methods for differentiating these cells to functional neurons of different classes or to overexpress mutant genes in the hESCs to model human disease (Marchetto et al., 2010b; Thomson et al., 1998). In addition, prior to the development of iPSC technology, somatic cell nuclear transfer (SCNT) was being applied in rare cases to study specific diseases (Rideout et al., 2002). However, soon after human cells were first reprogrammed (Takahashi et al., 2007), the modeling of neurodevelopmental and neurodegenerative diseases began in earnest, and the subsequent necessary effort to develop reliable protocols for differentiating the immature stem cells has progressed ever since. Neurogenetic disorders were modeled first (Dimos et al., 2008; Lee et al., 2009; Marchetto et al., 2010a; Zhang et al., 2010), followed by a few examples of sporadic and complex disorders (e.g., schizophrenia [SCHZ];
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Perspective Brennand et al., 2011; Paulsen et al., 2012; Pedrosa et al., 2011). While these modeling efforts are quite recent, concerns remain about the ability of reprogrammed fibroblasts to recapitulate disease phenotypes. Specifically, inadequate neuronal maturation, synaptic deficiency, and failed connectivity have been observed in many of the early-onset and neurodevelopmental diseases modeled so far (examples: familial dysautonomia [FD] [Lee et al., 2009], Rett syndrome [RTT] [Marchetto et al., 2010a; Ricciardi et al., 2012], Huntington’s disease [HD] [Chae et al., 2012], and SCHZ [Brennand et al., 2011]). It is possible that the apparent detection of synaptic deficits is partly the result of the types of measurements focused on so far. In neurodegenerative diseases and proteopathies, neuronal toxicity due to increased sensitivity to oxidative damage and proteasome inhibition seems to be more prevalent than strictly synaptic deficits. Examples include amyotrophic lateral sclerosis (ALS) (Mitne-Neto et al., 2011), Parkinson’s disease (PD) (Nguyen et al., 2011), Alzheimer’s disease (AD) (Israel et al., 2012), and Down syndrome, which mimics some aspects of AD (Shi et al., 2012). As the number of patients and types of neurological diseases being modeled increase, new patterns will emerge that could aid in developing earlier diagnostics tools and facilitate effective drug design. Significant interest among clinicians and the pharmaceutical industries has arisen as other neurological conditions are proposed to be modeled using iPSCs. Attractive candidate diseases include but are not restricted to bipolar disorder, major depression, multiple sclerosis, and idiopathic autism. Major Challenges in Modeling Neurological and Psychiatric Disease and Tools for Addressing Them When developing in vitro models, the main goal is to establish a meaningful parallel between the phenotypes observed in the dish and the disease pathology observed in vivo. An important set of challenges that currently surround this field involves the variability between clones and changes in clone genome and phenotype over passage and time. Targeted genome modification of hIPSCs using engineered constructs like zinc-finger nucleases (ZFNs) (Kim et al., 1996; Porteus, 2010), transcription activator-like effector nucleases (TALENs) (Bedell et al., 2012; Christian et al., 2010) and, more recently, clustered regularly interspaced palindromic repeats/CRISPR-associated (CRISPR/ Cas) system (Mali et al., 2013; Wiedenheft et al., 2012) present promising strategies for modeling monogenic and genetically defined disorders with reduced variability by generating isogenic control lines harboring defined genetic alterations (Soldner et al., 2011). For modeling sporadic diseases or complex neuropsychiatric disorders where there is no clear genetic etiology, the value of these targeted genomic approaches is less clear but still likely important. It is conceivable that identifying protocols that generate lineage-specific cells will solve this problem by allowing investigators to monitor the differentiation process more specifically. Defining and consistently obtaining the diseaserelevant neural cells at comparable levels of maturation should greatly reduce the phenotypic variability and highlight pertinent disease characteristics. Assessing neuronal network connectivity formation is important for understanding neuronal communication imbalance in disease but can be a challenging task because, as a general rule, the right subtype of neurons and the specific maturation time are not represented in the dish at
appropriate levels. To that end, designing cell-type-specific promoters may help in generating the desired populations of neurons that are directly involved in the disease being studied (for example, Hb9-positive cells for diseases involving alpha motor neurons such as ALS [Marchetto et al., 2008]). Additionally, single-cell expression profiling should further clarify the levels of population heterogeneity within in vitro cultures, and advances in media culture platforms and automated cell processing should provide the desired accuracy and consistency that will be required. For a number of neurological diseases, it remains unclear whether the phenotypes involved in the pathology are restricted to the neuronal population and to what extent the neighboring cells are also playing a major role. Improving the protocols for generation of cells present in the neuronal niche (i.e., astrocytes, oligodendrocytes, microglia, and endothelial cells) could reveal important disease phenotypes and contribute to the development of alternative therapies. Refining the techniques to analyze neuronal phenotypes will also help to detect more subtle differences. The field is driving strong interdisciplinary collaboration, bringing together technological advancements from multiple areas like electrical and mechanical engineering with principles of neuroscience and stem cell biology. New engineering and automation techniques are being applied to these types of studies as both engineers and the biotech industry and Big Pharma begin to explore and exploit this technology. Finally, we posit that many of the challenges facing disease modeling arise from the overall strategy employed. Many of the current disease modeling studies search for differences in gene expression generally or for basic functions that can be measured in vitro, i.e., functions that have been hypothesized to be correlated causally in the disease. Often these studies are not hypothesis driven but rather depend on existing techniques and the availability of somatic cells from whatever patients are available to the researcher. Researchers are beginning to work more closely with the clinicians who attend to and treat the patients to better understand the diversity of each of the patient populations to be studied and to obtain more restricted populations of patients (e.g., discordant monozygotic twins, drug-responsive versus nonresponsive cohorts, and severity of the disease). These kinds of collaborations between bench and bedside may not only lead to more targeted hypotheses but may also assist in decreasing the variability reported for in vitro modeling. While engineering platforms allow the researcher precision and control over the cellular microenvironment, in vivo transplantation of stem cell-derived populations of human pluripotent stem cells (hPSCs) and neurons into animal models presents a useful way to study human development and to model disease. Grafting NPCs at appropriate developmental stages could potentially utilize the myriad biochemical and biophysical cues provided in the endogenous niches to generate mature and functional populations of the desired cells. An excellent example is the transplantation of hPSC-derived forebrain NPCs into the neonatal mouse brain to generate cortical neurons with specific axonal projections and dendritic patterns corresponding to the native cortical neuron population (Espuny-Camacho et al., 2013). In addition, transplantation of hPSC-derived medial Neuron 80, October 30, 2013 ª2013 Elsevier Inc. 595
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Perspective ganglionic eminence (MGE) progenitors into the rodent brain produced GABAergic interneurons with mature physiological properties along an intrinsic timeline that mimics the endogenous human neural development (Nicholas et al., 2013). This emerging sector of stem cell biology has brought basic cell and molecular biologists together with engineers, clinicians, and large and small biotech companies. The new model organism is the human, and while this is a new field with plenty of caveats and unknowns, it is likely to stay around for the foreseeable future (Lancaster et al., 2013). IV. Applications of NSC to Disease and Therapy Human NSC Transplantation in Clinical Trials The discovery of the existence of NSCs throughout life in animals and then in humans led to rapid recognition of the therapeutic potential of these cells. Fetal human NSCs are an abundant source of neurons, astrocytes, and oligodendrocytes, with potential application to a variety of neurological conditions. Companies that were formed around the concept of transplanting human NSCs began the groundbreaking work of making clinical trials possible. Stem Cells, Inc. paved the way, generating clinical-grade banks of purified, fetal-derived human NSCs that are currently in use in clinical trials. They are being tested in patients with Pelizaeus-Merzbacher disease, a demyelinating condition of children that results in neurological dysfunction and death; the 2-year follow-up report indicates safety and improved and long-term myelination. These cells are also being tested in phase I/II clinical trials for spinal cord injury and the retinal disease dry age-related macular degeneration (AMD). In the latter case, human NSCs are not contemplated to replace the retinal pigment epithelial (RPE) cells that degenerate in AMD, as they do not generate that specific lineage but rather to substitute key RPE functions such as cytokine production and phagocytosis. Others pursuing the clinical application of human fetal NSCs include NeuralStem and the Azienda Ospedaliera Santa Maria, Terni, Italy. Both organizations are pioneering human fetal NSC transplants for ALS patients. Although it is early days, results thus far using well-defined human NSC products indicate that they can be transplanted safely and will integrate and generate long-lived progeny in their host. In contrast, the shocking report of tumor formation seen in a young Ataxia Telangiectasia patient given multiple mixed fetal human CNS grafts (Amariglio et al., 2009) cautions against the use of these cells outside of a clinical trial; furthermore, the disease indication should be carefully considered and tested in appropriate animal models to provide preliminary proof of concept and safety data before moving into humans. Given the rapid progress in pluripotent stem cell production of different neural lineages, one might ask whether fetal human NSC transplantation will at some point be superseded. The answer will depend on the relative safety profile of these different cell products and their ability to integrate and mature appropriately to provide efficacy. The next two decades will be revealing in this regard, and progress will be eagerly watched by patients and families afflicted by neurological disorders that could benefit from such transplants. Emphasis should be on performing well-designed clinical trials, and the NSC field must help educate patients to reduce the trafficking of unproven therapies. 596 Neuron 80, October 30, 2013 ª2013 Elsevier Inc.
PharmaNutri-Neurogenesis? NSCs exist throughout life in the hippocampal DG, but human VZ-SVZ stem cells stop actively generating neurons at about 2 years of age (Sanai et al., 2011). Adult hippocampal NSCs have life-long activity but their numbers decline in aging and are dramatically reduced in AD (Haughey et al., 2002), contributing to learning and memory deficits. Accumulated evidence that adult NSCs are responsive to environmental stimuli suggests ways to protect the activity of these cells, changing behavior and nutrition to enhance life-long NSC health. Psychiatric drugs can impact adult neurogenesis (Malberg et al., 2000), and small molecules could be designed more specifically for this target cell. Screening drugs for an impact on NSC function can help reduce toxicity and negative effects, such as the ‘‘chemobrain’’ side effects of some anticancer drugs (ElBeltagy et al., 2010). Growth factors that maintain NSC function are being explored as supplements, including infusion of IGF1, FGF, growth hormone, melatonin, and the BMP inhibitor Noggin (Bonaguidi et al., 2008), and the dramatic revitalization of aged hippocampal function demonstrated, for example, by loss of the Wnt inhibitor DKK1 (Seib et al., 2013) suggests that this will be a promising area for future translation. Utilizing Neural Progenitor Heterogeneity One particularly impressive example of the benefit of understanding progenitor heterogeneity is the progress made in treating childhood brain cancers. Comparison of in-depth gene expression analysis of normal murine progenitor cells and gene expression analyses of pediatric brain tumors has enabled the subdivision of medulloblastoma and ependymoma into different classes (Gibson et al., 2010; Johnson et al., 2010), providing more targeted therapies with better outcomes. In the future, a more complete understanding of human CNS progenitor subclasses will help to identify the cells responsible for different facets of neurological diseases and to target cell subsets more precisely to either enhance or diminish a particular cell group. Clinical Applications of Progenitor Cell Migration The long migrations of NSC progeny in vivo demonstrate that CNS cells have an astonishing capability to move about the nervous system. Receptor cytokines such as CXCR4-SDF1 guide normal migration and can be activated after trauma, e.g., in ischemic conditions, drawing cells out of adult germinal zones toward injured sites (Robin et al., 2006). While normal SVZ cells do not survive after attraction to these ischemic locations, it is possible that utilizing such homing mechanisms will help target therapeutic cells. One ongoing clinical trial utilizes the ability of immortalized NPCs to home to tumor sites; the cells are engineered to secrete a product that is activated once the cells reach glioblastoma lesions (Aboody et al., 2013). The powerful migratory ability of ventral forebrain-derived GABAergic neurons is being explored in translational studies that aim to deliver these cells therapeutically, anticipating that they will migrate and incorporate to dampen hyperexcitable states, for example, in epilepsy and spinal neuropathic pain (Bra´z et al., 2012; Maisano et al., 2009). One of the most astonishing examples of human NSC progeny migration is illustrated by human glial restricted precursors. When implanted into the shiverer demyelination mouse model, these cells produced oligodendrocytes that spread
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Perspective throughout the nervous system and essentially replaced the murine with human myelin (Windrem et al., 2008). Harnessing the mechanisms of neural migration will be valuable for more precise cell targeting and also to inhibit brain tumor-initiating stem cell spread. Enter Pluripotent Stem Cell-Derived NSCs Over the past 10 years, researchers have shown that the morphogenic gradients conceptualized and demonstrated in animal neurodevelopment are evolutionarily maintained and applicable to drive differentiation of diverse neural progeny from hPSCs. Stellar examples are the production of spinal motor neurons (Dimos et al., 2008; Wichterle et al., 2002) and midbrain substantia nigra dopaminergic (dA) neurons (Kriks et al., 2011). The story behind production of the latter cells underscores how critical it is to know the normal ontogeny. Dopaminergic cells were first derived from hPSCs by producing forebrain progenitors and then repatterning them to midbrain, but the resulting neurons largely lacked key markers of the midbrain A9 location desired for PD applications and were found to be unstable after transplantation into animal models. Instead, reiteration of the normal developmental track discovered in mouse, by first producing midbrain floor plate cells using high concentrations of SHH and then differentiating these into neurons, produced significantly more A9 dA neurons that were then shown to be stable and efficacious in animal models, providing the key proof of concept to move toward a clinical trial (Fasano et al., 2010; Kriks et al., 2011). While protocols for producing a diverse array of neural cells, both central and peripheral, are being developed, production of retinal cells has advanced, and subretinal transplantation of hESC-derived RPE cells for blindness disorders such as AMD is already in early phase clinical trials. The ability to produce a wealth of cell types from hPSCs for cell replacement and other potential therapies such as growth factors or drug delivery is undoubtedly exciting. One outstanding issue that has already been discussed, but worth noting again in this context, is that we still need to understand how to mature human NSC products appropriately. Our current approaches, which recapitulate early developmental morphogenic stimuli, are excellent at making early-stage cells, but many of the cell products remain immature, indicating that signals that normally extend development are lacking. Defining the stages and processes along the maturation arc is important, as these impact the ability of cells to integrate, migrate, and properly adopt desired characteristics, as seen for oligodendrocyte and photoreceptor transplantation (MacLaren et al., 2006; Warrington et al., 1993). If pluripotent cell-derived products are proven stable, safe, and efficacious when implanted into the human CNS, we can envision increasingly complex transplants, including not just RPEs but also photoreceptors, organoid-derived neural structures, or even 3D, tissue-printed personalized tissue grafts. The combination of engineering and stem cell research is powerful. Neural tissues that can self-assemble or be engineered from stem cells (Aoki et al., 2009; Eiraku et al., 2011; Kawamorita et al., 2002; Lancaster et al., 2013; Meyer et al., 2011) have a promising future to model complex, multicell neural diseases and as a basis for toxicity testing and mechanistic studies. The enormous advantage of having human neural cells widely avail-
able is something we could only dream about 25 years ago, and we predict that they will prove even more valuable and will likely supplant animal testing in efficacy studies, which have failed to model many human diseases; however, there is much work ahead to achieve this worthwhile goal. Given the rapid upward trajectory of biotechnological and biomedical advances, we can afford to let our imaginations range: will biological devices that incorporate cells and materials be developed, for example, as retinal prostheses or treatments for epilepsy or PD? Will we be protected by bioengineered sensors that use neural and computer elements, a ‘‘canary on a chip,’’ to detect stroke or external toxins? The future for the next generation of NSC researchers and for NSC translation is bright. Conclusion Thanks to great strides in our ability to observe and study germinal cells, and to investigate how neurons and glia are generated at cellular and molecular levels, we now have an impressive body of knowledge concerning NSC biology. Many of the foundational problems concerning NSCs were soluble only after a specific tool was developed (Table 1) and, with the extraordinary blossoming of technologies that is currently ongoing (Table 2), much more information is anticipated concerning the wealth of NSC types and their regulation. As imaging technologies advance, we should make significant headway in understanding how NSCs behave within endogenous niches and after implantation in vivo. Animal studies, notably in mouse, will continue to provide pioneering advances, especially to test application of new tools, but increasingly, we see the field moving toward pursuing the study of human NSC biology. The astonishing success of reprogramming somatic cells into neuronal and glial progeny with just a handful of genes (Najm et al., 2013; Vierbuchen et al., 2010) has made almost any cellular change seem possible, and the more we know about how NSCs tick, the better chance we have to produce, on demand, bona fide human neurons and glia for a multitude of in vivo and ex vivo applications. Overall, it has been inspiring to witness the extraordinary growth of knowledge in this area and to contribute to what is now an established field of NSC research, with great potential for advancing our understanding and healing of that most intricate organ, the nervous system. ACKNOWLEDGMENTS We would like to thank Qingjie Wang, Mary Lynn Gage, and Carol Marchetto for help in preparing the manuscript. F.H.G. is a founder for Stem Cells, Inc. and a member of the Scientific Advisory Board. S.T. is a founder of StemCulture, Inc. REFERENCES Aboody, K.S., Najbauer, J., Metz, M.Z., D’Apuzzo, M., Gutova, M., Annala, A.J., Synold, T.W., Couture, L.A., Blanchard, S., Moats, R.A., et al. (2013). Neural stem cell-mediated enzyme/prodrug therapy for glioma: preclinical studies. Sci. Transl. Med. 5, 84ra59. Aimone, J.B., Wiles, J., and Gage, F.H. (2009). Computational influence of adult neurogenesis on memory encoding. Neuron 61, 187–202. Altman, J. (1962). Are new neurons formed in the brains of adult mammals? Science 135, 1127–1128.
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