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Making cortex in a dish In vitro corticopoiesis from embryonic stem cells
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Nicolas Gaspard,1 Afsaneh Gaillard2 and Pierre Vanderhaeghen1,* 1Université
Libre de Bruxelles; IRIBHM (Institute for Interdisciplinary Research); Campus Erasme; Brussels, Belgium; 2CNRS and Poitiers University; Poitiers, France
Key words: embryonic stem cells, cerebral cortex, neuronal specification, temporal patterning, brain development, cerebral grafting
The cerebral cortex is arguably the most complex structure in the mammalian brain. It develops through the coordinated generation of dozens of neuronal subtypes, but the mechanisms involved in this daunting process of cell diversification remain poorly understood. We recently described a novel pathway by which mouse embryonic stem (ES) cells, cultured in the absence of any added morphogen but in the presence of a Sonic Hedgehog inhibitor, can recapitulate the major milestones of cortical development observed in vivo. In this system cortical-like progenitors seem to follow an intrinsic pathway to generate a surprisingly diverse repertoire of neurons that display most salient features of bona fide cortical pyramidal neurons. When grafted into the cerebral cortex in vivo, these neuronal populations develop patterns of axonal projections highly similar to those of native cortical neurons. The discovery of intrinsic corticogenesis, from stem cells to cortical circuits, sheds new light on the mechanisms of neuronal specification, and may open new venues for the modelling of cortical development and diseases, and for the rational design of brain repair strategies.
Brain Complexity from Stem Cell Simplicity? The cerebral cortex is one of the most complex structure in the organism, composed of a wide diversity of neuronal subtypes that populate specific cortical layers and areas. Pyramidal neurons make 80% of all the neurons in the cortex and are generated from cortical progenitors in a specific temporal order. Each neuronal subtype is characterized by a specific combination of laminar position, morphology, marker expression and pattern of connectivity, so that dozens of different subtypes of pyramidal neurons populate the cortex. Despite recent progress,1 the understanding of the mechanisms involved in the specification of distinct pyramidal neuronal *Correspondence to: Pierre Vanderhaeghen; Universite Libre de Bruxelles; IRIBHM; 808 route de Lennik; ulb campus erasme; Brussels, Brussels 1070 Belgium; Tel.: +32.2.5554186; Email:
[email protected] Submitted: 06/06/09; Accepted: 06/15/09 Previously published online as a Cell Cycle E-publication: http://www.landesbioscience.com/journals/cc/article/9276
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populations has proven to be difficult to study in vivo, due to the histological, cellular and molecular complexity of the developing cortex. Would there be a way to make corticogenesis easier to study? One possibility could be offered by the use of embryonic stem (ES) cells. ES cells have emerged as a powerful tool for developmental biology, allowing the in vitro re-creation of events of organogenesis in controlled and reproducible conditions, as well as the directed differentiation of specific neuronal populations, such as spinal motor neurons2 or midbrain dopaminergic neurons.3 The ability for mouse ES cells to generate forebrain-like progenitors was reported previously, but the identity of the neurons generated had remained uncertain.4-6 Besides it had not been tested yet whether a complex brain structure such as the cortex, comprising a complex array of different types of neurons, could be generated in vitro from ES cells. Using purely in vitro protocols, we have shown recently that ES cells can be very efficiently differentiated into cortical progenitors and subsequently cortical pyramidal neurons, following similar pathways as in vivo.7 This was achieved by recapitulating in vitro some of the key early steps leading to forebrain and cortical development. Once started along this physiological pathway, the process of in vitro corticogenesis could proceed normally up to the latest stages of cortical neuron specification, following a time-dependent self organizing pathway. Here we will discuss these recent findings in relation to in vivo development and another recent report of ES cell-derived corticogenesis.8
Recapitulating the Early Regional Specification of the Forebrain In Vitro The cerebral cortex is formed in the telencephalon, the anterior-most part of the forebrain. Forebrain development is thought classically to emerge through an intrinsic pathway, with the anterior neural plate constituting the primitive identity of the central nervous system.9 To explore the possibility that forebrain identity might emerge during differentiation of ES cells in default conditions, as during in vivo embryogenesis, we adapted a method of neurogenesis from adherent monocultures of ES cells.10 ES cells were cultured at low density as adherent monocultures10 in a chemically defined default medium (DDM) devoid of serum
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or any morphogen, but allowing cell survival by insulin. In such conditions, the majority of ES cells are converted into Sox1- and nestin-positive neural progenitors, which themselves give rise to neurons and finally glia, following the same temporal sequence as in vivo. Interestingly, the vast majority of the ES-derived neural progenitors express markers of the dorsal and ventral forebrain. These data are fully consistent with the so-called neural default model.11 In this model, anterior neural (forebrain) identity is proposed to constitute a primitive identity in the early vertebrate embryo, while non-neural and caudal neural fates arise through the action of extracellular morphogens, such as members of the Wnt and BMP families and retinoic acid. Thus, in minimal conditions in vitro, ES cells seem to faithfully replicate the critical initial step of neural development, further suggesting that neural induction and forebrain specification rely mainly on an intrinsic program of differentiation rather than on a complex interaction of extracellular cues.11 Of course our data do not rule out the role, perhaps permissive, of paracrine factors secreted by the ES cells themselves during neural differentiation, such a FGFs that were proposed previously to be involved in neural induction.12 Similarly it is conceivable that insulin present in the medium, acting through the IGF receptor pathway, may influence neural cell fate decision.13 However, it has been shown that neural induction from ES cells can happen in the absence of insulin and from ES cell lines where IGF signalling has been pharmacologically knocked down.14 Our findings are also well in line with recent reports showing preferential forebrain induction of mouse and human ES cells in the absence or inhibition of morphogen signals.15,16 Interestingly, several identities were found among forebrainlike ES-derived neural progenitors, corresponding to both ventral and dorsal telencephalon.7 The general mechanism of dorsoventral patterning is thought to be conserved along the neural tube and has been especially well studied in the spinal cord.17 It somehow resembles the mechanism of antero-posterior patterning, since gradients of morphogens induce specific expression of transcription factors in successive overlapping domains. The cross-regulatory networks created by these mutually repressive transcription factors then sharpen the boundaries between these domains. As in the spinal cord, Sonic hedgehog (Shh) is the key ventralizing morphogen of the forebrain and telencephalon (Fig. 1A), which promotes ventral identity at the expense of dorsal structures such as the cerebral cortex.18 We thus explored which signalling pathway were turned on during ES-forebrain induction and found that Shh was potently and precociously induced. To test whether, as in vivo, inhibition of Shh could promote dorsal forebrain, and thus potentially cortical fate, we took advantage of cyclopamine, a natural alkaloid known to inhibit potently and specifically the Shh pathways.19 When ES cells were cultured in DDM and cyclopamine at early stages of neural induction, most of the ventral forebrain progenitors could be respecified into dorsal progenitors, the majority expressing typical markers of the dorsal forebrain and cortical primordium (Fig. 1). We next examined the identity of the neurons generated in these conditions, using molecular markers of neurotransmission as well as electrophysiology. We found that progenitors differentiated www.landesbioscience.com
in the absence of cyclopamine give rise to a mixture of glutamatergic and GABAergic neurons, while addition of cyclopamine at the progenitor stage leads to the generation of population consisting largely of neurons expressing neuronal glutamate transporters and able to form glutamatergic synapses. In addition, these neurons display the typical unipolar and triangular shape of pyramidal neurons and, when grafted on top of cortical slices, show the ability to adopt a radial orientation, a specific feature of pyramidal neurons.20 These data are very reminiscent of the dorso-ventral patterning of the forebrain, where dorso-ventral identity of the progenitors is tightly linked to their competence to generate distinct types of neurons: ventral progenitors, specified through the action of Shh, generate mainly GABAergic neurons, while dorsal progenitors generate mainly pyramidal glutamatergic neurons.18
Temporal Generation of Layer-Specific Patterns of Neuronal Identity In Vitro While they all share common generic traits, cortical pyramidal neurons can be classified into many different subtypes, in particular according to which cortical layer they belong to (Fig. 1). Neurons residing in distinct cortical layers display specific patterns of axonal input and output, and this neuronal topology constitutes a major anatomical basis of cortical function. For instance, neurons residing in the deep layers project mainly to subcortical targets like the thalamus (layer VI), the midbrain or the spinal cord (layer V), whereas neurons of the upper layers II and III mainly connect with other cortical areas. Thanks to recent efforts aimed at identifying the molecular factors responsible for the specification of layerspecific patterns of projection,1 cortical neurons from different layers can be distinguished also according to the expression of specific molecular markers, the combination of which can define in a unique fashion the main populations of pyramidal neurons populating the various cortical layers (Fig. 1). Using these markers alone and in combination, we were able to determine that the pyramidal neurons generated from ES cells display a high level of diversity, with probably every cortical layer identity being represented. The majority of them express markers of layer V and VI neurons, like CTIP2, Tbr1 or FoxP2 and a small fraction of them express upper layer markers like Satb2 and Cux1. This neuronal diversity was further confirmed by in vivo grafting experiments, in which cortical-like neurons differentiated from ES cells were subsequently grafted in the frontal cortex of mouse neonates.7 A month following grafting, the axonal projections of the grafted neurons were examined, which revealed that ES-derived neurons send axonal projections to a whole array of typical brain targets of most cortical layers, including other cortical areas, thalamus and midbrain (Fig. 2). This surprising level of neuronal diversity leads to the question of the mechanisms underlying the generation of most types of cortical neurons from ES cells. Developmental studies have shown that laminar fate and subtype specification are linked to neuron birth-date, as early-born neurons settle in deep layers whereas late-born neurons populate the upper-layers.1 Cortical progenitors are thought to be at least in part multipotent and to undergo a
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Figure 1. (A) Schematic representation of the developing telencephalon, the anterior-most part of the forebrain, containing dorsal and ventral niches of neural progenitors that display specific competence to generate distinct types of neurons. Within the dorsal telencephalon, pyramidal neurons that populate distinct cortical layers express a specific combination of layer-specific markers. (B) Schematic representation of in vitro corticogenesis. ES cells cultured in a defined default medium DDM devoid of morphogens differentiate robustly into neural progenitors that display a forebrain-like identity. Dorsal forebrain identity can be induced further with the addition of Shh inhibitor cyclopamine, thereby allowing the generation of cortical progenitors. These cortical progenitors then spontaneously generate a wide array of different cortical neuronal types, through a progressive shift in competence similar to in vivo corticogenesis. 3
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Figure 2. (A and B) Schematic representation of the layer and area-specific patterns of connectivity of layer VI (A) and layer V (B) neurons, from motor (red), somatosensory (orange), auditory (green), and visual (blue) areas. (A) Layer VI neurons in all areas project mainly to the thalamic nuclei but show area-dependent intrathalamic specificity of connectivity: motor to the ventrolateral (VL) nucleus, somatosensory to the ventrobasal (VB) nucleus, auditory to the medial geniculate (MG) nucleus and visual to the lateral geniculate (LG) nucleus. (B) Layer V neurons in all areas project to more caudal structures and also show area-dependent specificity of connectivity: motor to the caudal pediculopontine nuclei (PPN) and the spinal cord, auditory to the inferior colliculus and visual to the superior colliculus and the rostral PPN. (C and D) When grafted in mouse neonatal cortex (C), ES-derived cortical neurons display a pattern of projections (D) corresponding to most cortical layers, but to highly specific areas, in particular visual (in blue) as well as limbic (not shown) identity. CC and LV in (C) stand for corpus callosum and lateral ventricle, respectively.
sequential shift in their competence to generate different subtypes of neurons, and such properties seem to be conserved at least in part when cortical progenitors are cultured in vitro.21 Remarkably, ES derived cortical progenitors show the same ability. In clonal conditions, single progenitors are able to generate different neuronal subtypes.7 Furthermore, the different neuronal subtypes are generated in the appropriate order both at the whole population level, as assessed by BrdU pulse-chase experiments, but also at the single progenitor level as further shown by clonal analyses:7 even when cultured at clonal density, the potential of progenitors evolves with time, as early progenitors mainly generate deep layer neurons and the progeny of late progenitors is shifted to upper layer neurons. Altogether, these data represent the first demonstration that the complex events leading to the generation of neurons displaying different layer-specific patterns of identity can take place outside of the developing brain and rely mainly on a cell population-intrinsic pathway. In addition, at least some aspect of the layered organization of the cortex can also be recreated in vitro: using a different approach, where ES cells are cultured as bowls of cells differentiating into www.landesbioscience.com
cortical-like progenitors, Eiraku and coll8 not only confirmed the sequential generation of distinct types of cortical neurons, but were also able to observe a striking polarized cellular organization, with neural progenitors occupying deeper layers of the bowls, and neurons accumulating at their periphery, following an organization highly reminiscent of a nascent cortical primordium. Altogether these data constitute a first proof of principle that a brain-like structure can emerge as a self-organizing cytoarchitecture in vitro, which constitutes a promising system to decipher some of the underlying mechanisms of cortical patterning. While in vitro systems of corticogenesis thus display remarkable similarities with in vivo developmental processes, it should be noted however that the differentiation taking place in vitro differs from in vivo corticogenesis in several important aspects. First, despite the fact that neurons with all six layers identities seem to be generated, one never sees the formation of a six-layered organization in vitro. Second, whereas in vivo deep and upper layer neurons each represent about half of the cortex, ES-derived pyramidal neurons are strongly skewed towards a deep layer identity.7,8 Interestingly, this is also the case when native cortical progenitors
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are grown in vitro,21 suggesting that some extrinsic cues, such as for instance interactions with incoming axons from the thalamus,22 are missing for the proper generation of the upper layer neurons. Alternatively, the cues instructive for particular neuronal types may be present but require a specific pattern of cytoarchitecture or polarity that may be less developed in a purely in vitro system. In this context it will be important to test whether features such distinct patterns of symmetric and asymmetric divisions, apicobasal polarity of neural progenitors, and interkinetic migration, can also be observed during in vitro corticogenesis, and how they might influence cortical neuron generation in vitro, quantitatively and qualitatively.
Specification of Cortical Areas Outside the Brain In addition to layers, the cerebral cortex can be subdivided into different functional areas. The motor, somatosensory, auditory, visual and limbic areas are composed of neurons sending modalityspecific axonal projections, that constitute a major landmark of areal identity (Fig. 2A and B). Surprisingly, in vivo grafting experiments revealed that ES-derived neurons seem to acquire mainly a limbic or visual area identity, corresponding essentially to the occipital (posterior) pole of the cortex (Fig. 2C). Indeed, ES-derived neurons grafted in the frontal cortex nonetheless send axonal projections to specific visual and limbic targets, like the lateral geniculate nucleus of the thalamus, the superior colliculus in the midbrain or the visual and limbic cortex itself. Importantly our results were all obtained with grafts in the frontal cortex, suggesting that the patterns observed were not due to the re-specification of the grafted neurons through the influence of the host.23-25 Confirming this hypothesis, when examining the molecular identity of ES-derived cortical progenitors and neurons before grafting, we found that most of them expressed typical markers of the occipital cortex, in particular CoupTFI/II transcription factors.7 These data thus indicate that progenitors and neurons generated in vitro in the ES cell system undergo an area-specific differentiation process that results in a surprisingly specific identity, corresponding mainly to occipital/ visual cortex. It remains to be determined by which mechanism cortical progenitors acquire such a specific areal identity. One possibility could be an effect of cyclopamine, as Shh pathways have been shown to interact with FGF8 signalling, which is a potent inducer of non-occipital cortical identities.26,27 Whatever the precise mechanisms involved, this intriguing observation suggests that the acquisition of cortical area identity can take place in vitro in the absence of external cues. While the development of cortical areas is thought to occur through the interplay of intrinsic and extrinsic mechanisms,25,28,29 these data constitute the first and surprising demonstration of the acquisition of cortical areal identity without any influence from the rest of the brain. These observations go well in line with previous reports using grafting of embryonic brain, that show that embryonic cortical progenitors are likely to be committed at an early stage to a specific areal identity.30 ES-derived corticogenesis combined with in vivo grafting will constitute a useful model to study crucial aspects of cortical areal 5
patterning that have remained controversial so far because of the complexity of the in vivo system.
An Ancestral Pathway Leading to Pyramidal Neuron Identity? While largely speculative at this stage, our data suggest that occipital areas may constitute a primitive identity, perhaps acquired early on during cortical evolution. Consistent with this view, cortical-like structures found in non-mammalian amniotes correspond mainly to visual-like structures, and primitive mammals mainly display sensory areas.31 In vitro corticogenesis could thus lead to an ancestral form of cortex already present in non-mammalian amniotes, which contains fewer neuronal layers and a less complex cytoarchitecture than the neocortex, i.e., the mammal-specific part of the cortex that is the most recent in evolution, with its unique six-layered structure and a large cohort of upper layer neurons. In this context it will be important in the future to study in vitro corticogenesis in other mammalian and non-mammalian species, to test further for the existence of an ancestral, evolutionarily conserved pathway of generation of pyramidal neurons.
Corticogenesis from Pluripotent Stem Cells: Perspectives for Models of Disease and Brain Repair Beyond its implications for our understanding of normal cortical development, in vitro corticopoiesis opens new venues to apply ES cell-based technology to cortical pathology, in particular for the modelling of cortical diseases, and the rational design of cortical neuron replacement therapies. Although using such a system for brain repair seems like a relatively distant perspective, future work should aim to test whether in vitro corticogenesis could be used to reconstruct lesioned cortical circuits in the adult brain, as suggested recently by the remarkable ability of native cortical progenitors to engraft and connect in the adult brain.32 Another exciting and more immediate possibility will be to apply such systems to induced human pluripotent stem (iPS) cells derived from patients presenting cortical diseases.33 This would provide a unique opportunity to model diseases of the cortex, such as specific forms of mental retardation, epilepsy, neurodegeneration, or even some neuropsychiatric diseases, for which existing animal models will always remain partially inadequate given the specific features of the normal and pathological human brain. In conclusion, the merge of embryonic stem cell technology and developmental neurobiology provides unprecedented opportunities to study one of the most complex structure in our brain, and could provide valuable tools to decipher the mechanisms underlying human brain development and diseases. Acknowledgements
The work of the authors presented here was funded by the Belgian FNRS/FRSM, the Belgian Queen Elizabeth Medical Foundation, the Simone et Pierre Clerdent Foundation, the Action de Recherches Concertées (ARC) Programs, the Interuniversity Attraction Poles Program (IUAP), Belgian State, Federal Office, the Walloon Region Excellence Program CIBLES, and the EU
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Marie-Curie Programme. P.V. is a Senior Research Associate of the FNRS, N.G. was funded as a Research Fellow of the FNRS. References 1. Molyneaux BJ, Arlotta P, Menezes JR, Macklis JD. Neuronal subtype specification in the cerebral cortex. Nat Rev Neurosci 2007; 8:427-37. 2. Wichterle H, Lieberam I, Porter JA, Jessell TM. Directed differentiation of embryonic stem cells into motor neurons. Cell 2002; 110:385-97. 3. Lee SH, Lumelsky N, Studer L, Auerbach JM, McKay RD. Efficient generation of midbrain and hindbrain neurons from mouse embryonic stem cells. Nat Biotechnol 2000; 18:675-9. 4. Bibel M, Richter J, Schrenk K, Tucker KL, Staiger V, Korte M, et al. Differentiation of mouse embryonic stem cells into a defined neuronal lineage. Nat Neurosci 2004; 7:1003-9. 5. Glaser T, Brustle O. Retinoic acid induction of ES-cell-derived neurons: the radial glia connection. Trends Neurosci 2005; 28:397-400. 6. Watanabe K, Kamiya D, Nishiyama A, Katayama T, Nozaki S, Kawasaki H, et al. Directed differentiation of telencephalic precursors from embryonic stem cells. Nat Neurosci 2005; 8:288-96. 7. Gaspard N, Bouschet T, Hourez R, Dimidschstein J, Naeije G, et al. An intrinsic mechanism of corticogenesis from embryonic stem cells. Nature 2008; 455:351-7. 8. Eiraku M, Watanabe K, Matsuo-Takasaki M, Kawada M, Yonemura S, et al. Selforganized formation of polarized cortical tissues from ESCs and its active manipulation by extrinsic signals. Cell Stem Cell 2008; 3:519-32. 9. Wilson SW, Houart C. Early steps in the development of the forebrain. Dev Cell 2004; 6:167-81. 10. Ying QL, Stavridis M, Griffiths D, Li M, Smith A. Conversion of embryonic stem cells into neuroectodermal precursors in adherent monoculture. Nat Biotechnol 2003; 21:183-6. 11. Levine AJ, Brivanlou AH. Proposal of a model of mammalian neural induction. Dev Biol 2007; 308:247-56. 12. Stern CD. Neural induction: 10 years on since the ‘default model’. Curr Opin Cell Biol 2006; 18:692-7. 13. Pera EM, Wessely O, Li SY, De Robertis EM. Neural and head induction by insulin-like growth factor signals. Dev Cell 2001; 1:655-65. 14. Smukler SR, Runciman SB, Xu S, van der Kooy D. Embryonic stem cells assume a primitive neural stem cell fate in the absence of extrinsic influences. J Cell Biol 2006; 172:79-90. 15. Wataya T, Ando S, Muguruma K, Ikeda H, Watanabe K, Eiraku M, et al. Minimization of exogenous signals in ES cell culture induces rostral hypothalamic differentiation. Proc Natl Acad Sci USA 2008; 105:11796-801. 16. Elkabetz Y, Panagiotakos G, Al SG, Socci ND, Tabar V, Studer L. Human ES cell-derived neural rosettes reveal a functionally distinct early neural stem cell stage. Genes Dev 2008; 22:152-65. 17. Jessell TM. Neuronal specification in the spinal cord: inductive signals and transcriptional codes. Nat Rev Genet 2000; 1:20-9. 18. Wilson SW, Rubenstein JL. Induction and dorsoventral patterning of the telencephalon. Neuron 2000; 28:641-51. 19. Chen JK, Taipale J, Cooper MK, Beachy PA. Inhibition of Hedgehog signaling by direct binding of cyclopamine to Smoothened. Genes Dev 2002; 16:2743-8. 20. Polleux F, Morrow T, Ghosh A. Semaphorin 3A is a chemoattractant for cortical apical dendrites. Nature 2000; 404:567-73. 21. Shen Q, Wang Y, Dimos JT, Fasano CA, Phoenix TN, Lemischka IR, et al. The timing of cortical neurogenesis is encoded within lineages of individual progenitor cells. Nat Neurosci 2006; 9:743-51. 22. Dehay C, Savatier P, Cortay V, Kennedy H. Cell cycle kinetics of neocortical precursors are influenced by embryonic thalamic axons. J Neurosci 2001; 21:201-14. 23. Pinaudeau C, Gaillard A, Roger M. Stage of specification of the spinal cord and tectal projections from cortical grafts. Eur J Neurosci 2000; 12:2486-96. 24. Barbe MF, Levitt P. Age-dependent specification of the corticocortical connections of cerebral grafts. J Neurosci 1995; 15:1819-34. 25. O’Leary DD, Chou SJ, Sahara S. Area patterning of the mammalian cortex. Neuron 2007; 56:252-69. 26. Rash BG, Grove EA. Patterning the dorsal telencephalon: a role for sonic hedgehog? J Neurosci 2007; 27:11595-603. 27. Hayhurst M, Gore BB, Tessier-Lavigne M, McConnell SK. Ongoing sonic hedgehog signaling is required for dorsal midline formation in the developing forebrain. Dev Neurobiol 2008; 68:83-100. 28. Vanderhaeghen P, Polleux F. Developmental mechanisms patterning thalamocortical projections: intrinsic, extrinsic and in between. Trends Neurosci 2004; 27:384-91. 29. Rash BG, Grove EA. Area and layer patterning in the developing cerebral cortex. Curr Opin Neurobiol 2006; 16:25-34. 30. Gaillard A, Roger M. Early commitment of embryonic neocortical cells to develop areaspecific thalamic connections. Cereb Cortex 2000; 10:443-53.
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