Self-organization of neural tissue architectures from pluripotent stem cells.

The Journal of Comparative Neurology Research in Systems Neuroscience DOI 10.1002/cne.23608

Review

Self-organisation of neural tissue architectures from pluripotent stem cells

Michael Karus1, Sandra Blaess1, Oliver Brüstle1 1

Institute of Reconstructive Neurobiology, University of Bonn LIFE&BRAIN

Center, and LIFE&BRAIN GmbH, 53127 Bonn, Germany

Abbreviated title: Neural tissue architectures Key words:

neural stem cell, rosette formation, cortical development, 3D culture

Corresponding author: Oliver Brüstle Institute of Reconstructive Neurobiology, University of Bonn 53127 Bonn, Germany Tel.: +49(0)228 6885 500 Fax.: +49(0)228 6885 501 Email: [email protected]

Grant support: The work was supported by the German Federal Ministry for Education and Research (BMBF; grants 01ZX1314A, 13N11830), the European Union (NeurostemcellRepair HEALTHF4-2013-602278; Eurostemcell HEALTH-F1-2010-241878; ScreenTox Grant Agreement No. 266753) and by a collaborative funding scheme between the California Institute of Regenerative Medicine and the BMBF (RFA 10-01 CIRM Early Translational II Research Awards; Grant 0316020, VDI: 1316020).

This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as an ‘Accepted Article’, doi: 10.1002/cne.23608 © 2014 Wiley Periodicals, Inc. Received: Mar 31, 2014; Revised: Apr 09, 2014; Accepted: Apr 09, 2014

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Journal of Comparative Neurology

Abstract (158 words) Although being subject of intensive research, the mechanisms underlying the formation of neural tissue architectures during development of the central nervous system remain largely enigmatic. So far, studies into neural pattern formation have been mainly restricted to animal experiments. With the advent of pluripotent stem cells it has become possible to explore early steps of nervous system development in vitro. These studies have unravelled a remarkable propensity of primitive neural cells to self-organise into primitive patterns such as neural tube-like rosettes in vitro. Data from more advanced 3D culture systems indicate that this intrinsic propensity for self-organisation can even extend to the formation of complex architectures such as a multilayered cortical neuroepithelium or an entire optic cup. These novel experimental paradigms not only demonstrate the enormous self-organisation capacity of neural stem cells, they also provide exciting prospects for studying the earliest steps of human neural tissue development and the pathogenesis of brain malformations in reductionist in vitro paradigms.

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(5555 words) 1. Introduction Self-organisation of single cells into complex architectures is a fundamental principle of all living matter. Even bacterial colonies can generate intricate growth patterns in response to chemotactic factors, which are present in their environment, or which they generate themselves from distinct substrates. For example, Escherichia coli bacteria spotted in the center of a Petri dish spontaneously grow in radial swarm-ring or lattice-like formations depending on the concentration of succinate in the agar (Budrene and Berg, 1995). In multicellular organisms, pattern formation provides the foundation for the development of highly complex tissues. During generation of the nervous system, billions of cells arrange themselves in a spatially and temporally highly coordinated manner. Moreover, and in contrast to the above described pattern formation by protozoa, the cells undergoing the intricate process of tissue formation have to dynamically change their characteristics as development proceeds (e.g. they develop from a neural precursor into a fully matured neuron). For instance, during development of the neocortex, the competence of cortical progenitors to generate different types of projection neurons changes over time. This temporally ordered generation of projection neurons is a prerequisite for the formation of the six layered tissue architecture of the neocortex (Desai and McConnell, 2000; McConnell, 1988; Shen et al., 2006). Segregation of immature and differentiating neural cells into distinct groups and architectures is a long recognized phenomenon. Such 'sorting out' processes have been extensively analysed in classical fetal cell reaggregation and transplantation paradigms and are considered to be of major importance for the development of tissue architectures (Ajioka and Nakajima, 2005; DeLong, 1970; Krushel and van der Kooy, 1993; McLoon et al., 1982; Steinberg and Gilbert, 2004; Whitesides and LaMantia, 1995). For example, Nose and colleagues demonstrated that mixed suspensions of cells 3 & Sons John Wiley

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expressing either P-cadherin or E-cadherin form discrete cell aggregates based on their respective cadherin expression (Nose et al., 1988). Although frequently addressed in the context of developmental studies, selforganisation has, so far, received only limited attention in the stem cell field. This is somewhat remarkable, considering that neural stem cells (NSCs) derived from both embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) spontaneously form prominent rosette structures reminiscent of the developing neural tube even in 2D culture conditions. More recent studies have demonstrated that prolonged cultivation under differentiating conditions can prompt NSCs to form region-specific neural histo-architectures including a stratified cortical neuroepithelium and an entire optic cup. These structures exhibit remarkable similarities to bona fide cortical and retinal tissue, respectively. In this review we revisit recent insights into the generation of neural tissue architectures in pluripotent stem cell (PSC)-derived NSC cultures. We compare the ability of different PSC-derived NSC populations to engage in pattern formation in both adherent and suspension culture paradigms. In the context of suspension culture we also highlight the capability of self-organised neural tissue to undergo complex tissue-movements, leading to the formation of e.g. primitive optic cups. 2. Neural tissue architecture in adherent culture The neural rosette as a lineage-specific primitive architecture Under appropriate conditions mammalian PSCs efficiently differentiate into the neural lineage in vitro. The classic two-step neural induction protocol consists of an embryoid body (EB) formation phase followed by plating of the EBs on an adhesive substrate. Subsequently, emerging NSCs are kept in a proliferative state in the presence of fibroblast growth factor 2 (FGF2). Numerous studies employing such a differentiation paradigm have reported on the emergence of epithelial cells, which express the NSC marker nestin and display an elongated and columnar shape. These cells are arranged in a 4 & Sons John Wiley


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blossom-like fashion and start forming neural tube-like structures called neural rosettes (Fig. 1A). Such observations have first been made using murine ESCs (Okabe et al., 1996) and were later recapitulated in human ESC cultures (Zhang et al., 2001). Interestingly, several years earlier Tomooka and colleagues had already reported on the appearance of neural tube-like structures in cultures of primary neural cells isolated from embryonic day 9-10 mouse embryos (Tomooka et al., 1993). Importantly, the ability of neural cells to form rosette structures is not an in vitro artefact resulting from NSC culture conditions. Rosette formation has also been documented after transplantation of PSC-derived NSCs into the rodent brain (Brüstle et al., 1997). Even human brain tumors, in which neural stem cell programs might have been reactivated, contain neural rosettes (Brustle et al., 1992; Cruz-Sanchez et al., 1991; Eberhart et al., 2000; Hsu et al., 2012; Satomi et al., 2011). Thus, with respect to pattern formation in neural tissues, the neural rosette can be regarded as a primitive architecture in the neural lineage. In 2008 Elkabetz and colleagues published a seminal article, in which they extensively characterized primitive rosette-forming NSCs (R-NSCs) derived from human ESCs (Fig. 1A). R-NSCs can be identified by the expression of transcription factors such as SOX1 (SRY-box containing gene 1), SOX2, and PAX6 (paired box gene 6). These cells exhibit a default anterior neural plate identity and are amenable to patterning using defined concentrations of potent morphogens.

Importantly,

NSCs

with

rosette-forming

abilities

and

characteristics similar to R-NSCs exist in vivo: such cells have successfully been isolated from the murine anterior neural plate (Elkabetz et al., 2008). Upon further expansion in medium containing FGF2 and epidermal growth factor (EGF), R-NSCs lose their spatial organisation and instead grow in a seemingly random fashion. This late stage cell population has been named NSCFGF2/EGF (Elkabetz et al., 2008) (Fig. 1A, B). The expression profile of NSCFGF2/EGF and their differentiation capacity are comparable to the radial glia-like phenotype observed in a fetal NSC population that has been previously described by Conti and colleagues (Conti and Cattaneo, 2010; Conti et al., 2005).

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In neural rosettes composed of R-NSCs the cell adhesion molecule NCadherin (N-CDH), the tight junction protein Zonula occludens 1 (ZO-1) and the glycoprotein prominin (CD133) are localised at the core of the rosette structure (Elkabetz et al., 2008; Pankratz et al., 2007) (Fig. 1A). Interestingly, these proteins are typically found at the apical membrane of the developing neuroepithelium in vivo (Götz and Huttner, 2005). Thus, the restriction of such molecules to the inner part of the rosette structure suggests an apico-basal polarity of the cells within the rosette. Moreover, mitotic phospho-Histone H3 (pHH3)-positive cells are usually found at the centre of the rosette structure (Fig. 1A), although the majority of rosette cells express the cell cycle marker Ki67 and can be labeled using bromodeoxyuridine (BrdU) (Elkabetz et al., 2008). The distribution of pHH3-positive cells suggests that R-NSCs within the rosette may undergo interkinetic nuclear migration, a process in which the nucleus of a cell periodically moves between the apical and basal membrane during the cell cycle. Indeed, a phenomenon reminiscent of interkinetic nuclear migration has recently been documented in neural rosettes using time-lapse analysis (Falk et al., 2012; Nasu et al., 2012). Strikingly, neural crest (NC) cells, which give rise to the peripheral nervous system and develop at the dorsal edge of the neural tube in vivo, are commonly found at the periphery of neural rosette structures (Lee et al., 2007; Mica et al., 2013), suggesting that the spatial organisation between CNS and NC cells is maintained in vitro (Chizhikov and Millen, 2005; Krispin et al., 2010). However, it is not clear, whether this topographic organisation is due to cellular delamination processes of NC cells from early rosette structures or rather reflects a simple cellular sorting-out phenomenon during in vitro culture. In an attempt to derive a proliferative primitive NSC population from human PSCs, Koch and colleagues generated yet a second type of rosette forming NSCs. Such NSCs also express PAX6, SOX2, and nestin (Fig. 1C) and stably proliferate in the presence of FGF2, EGF, and low concentrations of the cell culture supplement B27. Even upon extensive long-term culture (up to 75 passages) they retain a high neurogenic capacity and hence have been named long-term neuroepithelial stem cells (lt-NES cells) (Falk et al., 2012; Koch et al., 2009). Interestingly, lt-NES cells exhibit a posterior identity 6 & Sons John Wiley


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compatible with a ventral anterior hindbrain fate. This regional bias might be due to the posteriorising effect of FGF2 (Stern, 2001). Nevertheless, lt-NES cells can still be instructed to acquire adjacent regional fates such as midbrain dopamine neuron-like cells and spinal motoneurons (Koch et al., 2009). Thus, lt-NES cells are more plastic than previously described NSC populations. In terms of neural tissue architecture, lt-NES cells self-assemble into small rosette

structures,

which

are

characterised

by

prominent

ZO-1

immunoreactivity at the centre of the rosettes (Fig. 1A, C). As opposed to RNSC-derived rosettes, lt-NES-generated rosettes are considerably smaller and often composed of only a few cells. Importantly, lt-NES cells do not represent an artefact resulting during in vitro differentiation of PSCs. A recent study showed that a similar cell type can be derived from human fetal hindbrain cells (Carnegie stage 15-17). Such primary human NSCs stably proliferate under the culture conditions used for lt-NES cell propagation and retain their ability to generate small rosette structures for up to at least 60 passages (Tailor et al., 2013). Recently,

insights

into

basic

mechanisms

underlying

early

neural

differentiation have been implemented in vitro and have greatly facilitated the derivation of novel primitive NSC populations from PSCs. A major improvement in efficient NSC generation was achieved by inhibiting transforming growth factor β (TGFβ)/activin and bone morphogenetic protein (BMP) signaling during the early steps of neural induction (Chambers et al., 2009; Kim et al., 2010; Zhou et al., 2010). Both signaling pathways use SMAD (MAD homolog) proteins as downstream effector molecules, and the application of potent antagonists of the two signaling pathways is now generally referred to as "dual SMAD inhibition". Dual SMAD inhibition in combination with adherent culture conditions results in rapid and efficient differentiation of human PSCs into primitive neuroectodermal cells within only a few days (Chambers et al., 2009). Pharmacological modulation of key signaling pathways meanwhile has become an important concept for neural induction paradigms. More recently, two independent studies reported on the derivation of earlier human primitive neuroectodermal cells under adherent

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culture conditions. Li and colleagues stimulated canonical Wingless/Int-1 (WNT) as well as leukemia inhibitory factor (LIF) signaling and suppressed TGFβ/activin signaling, resulting in a stably expandable NSC population (Li et al., 2011). Reinhardt and colleagues first applied dual SMAD inbition for neural induction and subsequently used small molecules to activate WNTand SHH signaling to keep respective NSCs in a proliferative state (Reinhardt et al., 2013). Under both conditions primitive neuroepithelial cells proliferate in large colonies over a prolonged period of time. Interestingly, these primitive neuroepithelial cells do not readily form neural rosette structures and lack signs of polarization. Even though they express N-CDH and ZO-1 at the cell membrane, these proteins are not localized in a polarized fashion (Fig. 1A, B). Cultivation of these primitive neuroepithelial cells in medium containing high concentrations of FGF2 results in a rapid reorganisation into neural rosette structures (Li et al., 2011; Reinhardt et al., 2013). Thus, pre-rosette NSCs appear to represent a very primitive neural cell type. In line with this they can be instructed to adopt either a CNS or a NC fate. While these different NSC populations have been well characterized in recent years, their lineage relationship still remains unclear. It is tempting to speculate that pre-rosette NSCs, R-NSCs, lt-NES cells, and NSCFGF2/EGF represent different developmental stages along a linear differentiation route from PSCs to differentiated neurons and glia. In such a scenario, the ability to form neural rosettes would be restricted to defined intermediate stages along this route, i.e. to R-NSCs and lt-NES cells (Fig. 1A). However, it should be taken into consideration that these different NSC populations have distinct regional identities and that clear evidence for a lineage relationship between the different populations is yet to be provided. Recapitulating cortical development using PSC-derived NSCs The fact that NSCs can generate simple histo-architectures brought up the question, whether cell culture models could be used to mimic more complex developmental cascades such as the sequential generation of cortical layers. During the development of the mammalian neocortex neural precursor cells

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give rise to distinct types of glutamatergic projection neurons in a predetermined spatio-temporal fashion (for review see e.g. (Lui et al., 2011; Molyneaux et al., 2007). A graphical outline of this complex morphogenetic process is given in Fig. 2A. Initially, a single sheet of pseudostratified neuroepithelial cells (NECs) undergoes symmetric cell divisions to increase the size of the progenitor pool. With the onset of neurogenesis, these neuroepithelial cells begin to transform into radial glia cells (RGCs). RGCs reside in the ventricular zone but extend long processes from the ventricular wall to the pial surface. They divide asymmetrically and contribute to cortical neurogenesis by producing projection neurons either directly or by generating intermediate progenitors (IPs). IPs are positive for the transcription factor TBR2 (T box gene 2) and can generate cortical projection neurons via symmetric cell division. A distinctive feature of the developing human brain is the generation of a large number of outer radial glia cells (oRGCs) from ventricular zone RGCs. ORGCs represent a third type of cortical progenitor and are thought to significantly contribute to the expansion of the neocortex during mammalian brain evolution (Hansen et al., 2010; LaMonica et al., 2013). They undergo self-renewing divisions and generate cortical neurons primarily through the production of IPs. Cortical progenitors initially produce deep layer neurons (layers V and VI) positive for the transcription factors TBR1 and CTIP2 (COUP TF1-interacting protein 2), followed by upper layer neurons (layers II-IV) expressing the transcription factors SATB2 (special ATrich sequence binding protein 2), BRN2 (Brain-specific homeobox/POU domain protein 2, also known as Pou3f2), and CUX1 (cut-like homeobox 1). Importantly, upper layer neurons need to migrate through the deep layer neurons to reach their correct destination. This migration is supported by early born layer I neurons, the so-called Cajal-Retzius cells (Bielle et al., 2005), which express TBR1 and the glycoprotein reelin. The latter acts as an important guidance cue for migrating postmitotic projection neurons. Upon neural induction, PSC-derived NSCs first acquire an anterior identity. In line with this, in vitro differentiation paradigms that do not include growth factors with posteriorizing effects allow for a direct differentiation of forebrain cell types. In mouse cells, such non-directed differentiation protocols lead to 9 & Sons John Wiley

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ventral forebrain identities, presumably due to intrinsic expression of the ventralising morphogen Shh (Gaspard et al., 2008). As a consequence, blockage of the Shh pathway by cyclopamine and subsequent differentiation of murine ESC-derived neural precursor cells result in the generation of different cortical neuronal subtypes in a sequence reminiscent of the sequential formation of layer-specific neurons in vivo (Gaspard et al., 2009; Gaspard et al., 2008). Upon grafting into the rodent brain such neurons exhibit morphologies similar to cortical projection neurons. Moreover, they were found to extend long projections into cortical and even subcortical areas in a layer- and area-specific manner (Gaspard et al., 2008; Ideguchi et al., 2010). Based on the observation that endogenous WNT signaling instructs a dorsal identity of human PCS-derived NSCs, it became possible to generate human cortical neurons in vitro (Li et al., 2009; Zeng et al., 2010). However, two important questions remained: First, do different classes of cortical progenitor cells (RGCs, IPs, and oRGCs) emerge with ongoing cultivation? Second, does the generation of human cortical neurons from PSC-derived neural precursor cells happens in a sequential manner? The first evidence that these aspects of cortical development can be recapitulated by PSC-derived neural precursor cells was provided by Shi and colleagues. They differentiated several hESC- and hiPSC lines into the neural lineage under chemically defined culture conditions. Neural rosettes readily emerged within a couple of days. Cells within the rosettes proliferated and displayed an apico-basal polarity evident in the apical localization of CD133, ZO-1, and N-CDH (Shi et al., 2012a; Shi et al., 2012b). Strikingly, while initially most pHH3-positive NSCs were confined to the inner part of the rosette structure, a small fraction of such cells was found at more basal positions with ongoing cultivation, reminiscent of the subventricular zone of the developing mammalian cortex. In line with this, TBR2-positive IPs as well as phospho-Vimentin-positive oRGClike cells bearing a single, basally oriented process have been observed in these neural tube-like structures (Shi et al., 2012b) (Fig. 2B). Such change of the precursor cell composition could suggest that neuronal differentiation occurs in a sequential manner. In fact the appearance of TBR2-positive IPs was accompanied by an emergence of immature neurons positive for the microtuble-associated protein doublecortin at the outer rim of the rosette 25

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days after neural induction. Further cultivation for up to 100 days resulted in the sequential generation of deep layer neurons (TBR1-/ CTIP2-positive) and upper layer neurons (SATB2-positive). However, although the generation of cortical neurons occurred in a sequential manner similar to normal development, the neurons in this 2D culture paradigm failed to arrange themselves in distinct neuronal layers reflecting an inside-out gradient of neurogenesis. Moreover, it remained unclear, whether the cortical progenitor cells and neurons segregated into primitive layers or whether both types of cells were intermingled (Shi et al., 2012b) (Fig. 2B). Nevertheless, these studies showed that cultivation of primitive neural rosettes with anterior forebrain character under differentiating conditions can mimic aspects of cortical development. 3. Neural tissue architecture in 3D suspension culture Classical neurosphere culture Even though neural self-organisation phenomena can be unequivocally observed in conventional monolayer cultures, suspension culture paradigms might be better suited for the spontaneous development of complex neural histo-architectures, since they enable three-dimensional growth. In fact, 3D culture conditions have been used in the neural stem cell field for many years. In the early 1990s the first protocols for suspension cultures of NSCs isolated from both the fetal and adult rodent brain were published. NSCs were grown as free-floating aggregates termed neurospheres that could be passaged over an extended period of time in the presence of EGF and FGF2. (Reynolds et al., 1992; Reynolds and Weiss, 1992; 1996). Since then the neurosphere culture system serves as a gold standard for the cultivation of mammalian NSCs, and its strengths and limitations have been extensively reviewed (Pastrana et al., 2011; Reynolds and Rietze, 2005). In terms of histoarchitecture, neurospheres generated from primary cells were initially considered to be rather primitive, with a heterogenous mixture of neural stem or progenitor cells and some postmitotic cells arranged in a random manner. Yet, several studies reported on a topographical segregation of neural stem/progenitor cells and differentiated cell types within a neurosphere


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depending on the regional origin of the primary tissue. In most cases nestinpositive neural stem/progenitor cells were primarily found at the edge of the neurosphere, while βIII-tubulin-positive neurons and glial cells expressing GFAP (glial fibrillary acidic protein) were located in the inner core of the neurosphere (Campos et al., 2004; Jensen and Parmar, 2006; Sirko et al., 2007; von Holst et al., 2006). However, in some cases proliferative and differentiated cells are arranged in a way, that the latter are primarily located at the outer rim (Sasaki et al., 2008; Sirko et al., 2007; Weible and Chan-Ling, 2007). Despite this primitive cellular separation, bona fide neural tissue architectures (e.g. a layered cortical neuroepithelium) do not form within neurospheres. This inability is in line with the current view that the neurosphere-forming cell is a radial glia-like stem cell (Karus et al., 2011; Pastrana et al., 2011; Reynolds and Rietze, 2005; Young et al., 2010), which may correspond to the NSCFGF2/EGF cell. As such the neurosphere-forming cell most likely represents a late stage neural precursor cell that lost the ability to engage into complex pattern formation. Suspension culture for the analysis of cortical development While the classical neurosphere culture system does not yield neural tissue architectures, PSC-based suspension cultures enable the generation of more complex tissue structures. In order to differentiate PSCs, they have traditionally been allowed to form EBs in the presence of fetal calf serum upon detachment from mouse embryonic fibroblast feeder layers. Such EBs usually contain derivatives of all germ layers, and hence do not represent an appropriate system to study the development of lineage-specific histoarchitecture. In that context it has been shown that medium, which was conditioned by hepatocellular carcinoma (HepG2) cells, instructs a primitive ectoderm-like fate of murine ESCs-derived EBs (Rathjen et al., 1999). In 2002 Rathjen and colleagues used HepG2 cell-conditioned medium in combination with classic EB-medium to facilitate neural induction from murine ESCs. Nine days after neural induction the resulting cell aggregates homogeneously developed an outer neuroepithelial layer composed of Sox1- and Sox2expressing cells, demonstrating that primitive neural tissue architectures can



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in principle develop from PSCs in a suspension culture paradigm (Rathjen et al., 2002). A major drawback of these early studies was the use of serumcontaining medium, which inevitably causes batch-to-batch variations and inconsistent experimental outcomes. Therefore, a standardisation of such cultures appears virtually impossible. In this context, the implementation of a serum-free suspension culture allowing

the

instruction

of

specific

regional

identities

(e.g.

dorsal

telencephalon) by adding potent morphogens represented an important improvement (Watanabe et al., 2005). To further standardize this so called SFEB protocol (serum-free floating culture of embryoid body-like aggregates) Eiraku and colleagues used ultra low-attachment 96-well plates for the quick reaggregation (SEFBq) of dissociated ESCs into an EB-like structure of a defined size (Eiraku et al., 2008). Further cultivation of the EBs in the presence of potent WNT- and TGFβ-inhibitors led to the formation of polarized cortical neuroepithelia from murine and human ESCs. Such neuroepithelia, reminiscent of small neural tube-like structures, were initially composed of cortical progenitor cells positive for PAX6 and the telencephalic marker FOXG1 (Forkhead box G1). These progenitor cells underwent interkinetic nuclear migration. Upon prolonged periods of culture (more than one week for mouse and more than six weeks for human neuroepithelia), TBR2-positive IPs emerged at the basal side of the neuroepithelia, indicating an early segregation into ventricular zone- and subventricular zone-like regions. Moreover, reelin-/TBR1-positive neurons could be observed at the outer rim of these neural tube-like epithelia. This demonstrated that a primitive layering of proliferative and postmitotic cortical cells occurs in this SFEBq culture paradigm. In an attempt to develop a further 3D culture system we have successfully adapted the adherent culture system presented by Shi et al. to a suspension-based culture regime (unpublished data). Under these conditions a similar primitive separation of proliferative and postmitotic cells generated from hiPSCs can be observed 50 days after neural induction (Fig. 3A-C), demonstrating the propensity of PSC-derived cortical cells to generally segregate into primitive layers in 3D culture paradigms. Using BrdU-based birth-dating in mouse ESC-derived cortical neuroepithelia, Eiraku and


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colleagues demonstrated that Cajal-Retzius cells, deep layer neurons and upper layer neurons were generated sequentially. However, although deep and upper layer neurons appeared to be spatially segregated, distinct neuronal layers reflecting an inside-out mode of neurogenesis were not discernible (Eiraku and Sasai, 2011; Eiraku et al., 2008; Nasu et al., 2012). Interestingly, a large-scale transcriptome analysis of hiPSC-derived cortical neuroepithelia generated along the SFEBq protocol and maintained for 50 days in vitro revealed a remarkable similarity of the transcriptome to that of human fetal cortical tissue at ten weeks after conception (Mariani et al., 2012). While these results indicate that in vitro generated and fetal cortical neuroepithelia have a similar gene expression program, the classical SFEBq culture seems not to be sufficient to generate cortical neuroepithelia exhibiting an advanced degree of stratification. Using a culture system reminiscent of the SFEBq culture, Lancaster and colleagues have recently generated so-called cerebral organoids (up to 4 mm in diameter) that contain large cortical neuroepithelia (Fig. 4). In their culture system PSCs are first aggregated in a multi-well plate. After neural induction these aggregates are embedded in a small matrigel droplet and subsequently transferred to a spinning bioreactor to enhance nutrient absorption (Lancaster et al., 2013). Strikingly, cortical neuroepithelia generated in this system display advanced neuronal layering. After 30 days in vitro an outer layer of TBR1- and reelin-positive neurons, which in vivo represent the earliest born neurons in the cortex, was clearly separated from a PAX6-positive germinative zone. After 75 days a zone of CTIP2-positive deep layer neurons and a more superficial layer of SATB2-positive upper layer neurons had formed (Fig. 5). These data suggest that late born (i.e. upper layer) neurons may have migrated through the layer of early born (i.e. deep layer) neurons recapitulating the migration processes observed during fetal cortical development. Another striking observation was that cortical neuroepithelia contained a remarkable number of SOX2-/phospho-vimentin-positive oRGCs forming an outer subventricular zone-like region, which was clearly separated from apical SOX2-positive RGCs. Since oRGCs were presumably able to undergo an asymmetric division as shown by the asymmetric distribution of



SOX2 in dividing cells, it can be assumed that oRGCs significantly contributed to neurogenesis in these cortical neuroepithelia. Remarkably, some of the cerebral organoids also contained a small band of cells expressing the cortical interneuron marker calretinin. Murine and human cortical interneurons originate not in the cortex, but in the ganglionic eminences in the ventral telencephalon, from where they migrate tangentially into the developing cortex (Hansen et al., 2013; Metin et al., 2006; Wonders and Anderson, 2006). Interestingly, the presence of calretinin-positive neurons in cerebral organoids in vitro was dependent on the overall presence of progenitor cells positive for the transcription factor NKX2-1 (NK2 homeobox 1), a marker for interneuron progenitors. This finding could suggest that regions of different positional identities may interact with each other in a single cerebral organoid. Of interest, in addition to cortical regions, cerebral organoids contained neural cell types reflecting other regional identities such as hippocampal-like regions, choroid plexus and primitive retina (Fig. 4). Although these observations highlight the ability of neural cells to spontaneously form a multitude of neural tissues with different regional identities, it might be advantageous to control for the formation of neural tissues with a particular regional identity. A recent article published by the Sasai group demonstrates that this is indeed possible. The authors improved the SFEBq system by adding chemically defined lipids and by culturing the cells in a high oxygen atmosphere. Using small molecules to initially block both canonical WNT- and TGFβ-signaling the authors were able to restrict the regional identity of hESC-derived neural cells primarily to cortical fates (Kadoshima et al., 2013). Strikingly, the authors frequently observed patterning along the anteroposterior axis of a single cortical neuroepithelium, based on their analysis of the transcription factors COUP-TF1 (also know as Nr2f1, nuclear receptor subfamily 2, group F, member 1) and SP8 (trans-acting transcription factor 8). Along the anteroposterior axis of the developing forebrain, COUP-TF1 is expressed in a low-to-high gradient while SP8 is expressed in a high-to-low gradient (Sansom and Livesey, 2009), expression patterns that were also observed in the in vitro-generated cortical neuroepithelia. Similar to cortical development in vivo, these expression gradients could be modulated by the addition of


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FGF8b. Moreover, addition of low amounts of a SHH agonist induced the formation of ventral NKX2-1-positive ganglionic eminence-like regions in close proximity to

PAX6-positive

presumptive

cortical

regions.

These

are

remarkable results, since they show that defined concentrations of morphogens can be applied in a hESC-derived 3D suspension culture to pattern the obtained neural tissue and to induce adjacent brain regions. In this context it will be interesting to further explore whether such spatially distinct regions within a 3D culture interact and whether ventral ganglionic eminencelike regions do indeed contribute interneurons to presumptive dorsal cortical regions. When Kadoshima et al. analysed layer formation in more detail at layer formation, they first observed the formation of an outer neuronal layer containing reelin-positive cells after 42 days in vitro. With ongoing cultivation the developing cortical neuroepithelia generated deep and upper layer neurons in an inside-out pattern. Importantly, deep layer neurons even expressed CaMKII-α (calcium/calmodulin-dependent protein kinase II alpha), a marker for mature neurons, after 112 days, suggesting an advanced neuronal maturation (Fig. 5). A similar level of complexity was documented for the germinative regions, which were split into a ventricular zone, a subventricular zone, and an intermediate zone containing RGCs, IPs, and oRGCs, respectively (Kadoshima et al., 2013). These observations indicate that hPSC-derived cortical neuroepithelia can develop into a complex stratified tissue that exhibits cardinal features of the developing human fetal cortex. Analysis of tissue movements in 3D suspension culture In the context of in vitro tissue development three different terms describing different mechanisms underlying self-organisation, have recently been introduced (Sasai, 2013a; b): self-assembly, self-patterning and self-driven morphogenesis. These terms can also be applied to describe the in vitro formation of neural tissues with different degrees of complexity. When pre-rosette NSCs are treated with FGF2, these uniform non-polar cells spontaneously rearrange their relative positions, resulting in a patterned structure (i.e. the neural rosette). Such phenomena can be categorized as



self-assembly. During the development of cortical neuroepithelia in vitro a seemingly homogeneous population of primitive cortical progenitor cells gives rise to a heterogeneous pool of different cell types, which form separate cellular layers in a distinct temporal sequence, a process that can be referred to as self-patterning. Both self-assembly and self-patterning largely relate to changes at a cellular level, leading to tissue self-organisation. In contrast, selfdriven morphogenesis takes into account that the generation of complex tissue architecture is also based on changes in the morphology of an already established tissue structure. Such ‘neural tissue movements’ have recently been described in the context of 3D cultures. For example, during the in vitro development of cortical neuroepithelia the apical side of the cortical progenitor cells is initially located at the surface of the cortical aggregates. In contrast the basal side is oriented towards the core of the aggregate. Yet, with ongoing cultivation, complex morphogenetic processes lead to the reorientation of the apicobasal cell polarity, yielding a continuous epithelium whose apical side is oriented inwards, while its basal side is oriented outwards (Kadoshima et al., 2013) (Fig. 6A). Another remarkable example of complex neural tissue movements in a 3D culture paradigm is the development of optic cups from murine and human PSCs (Eiraku et al., 2012; Eiraku et al., 2011; Nakano et al., 2012). During normal development of the optic cup, complex tissue movements are necessary for the separation of the neural retina from the retinal pigment epithelium (Graw, 2010). While the primitive retinal neuroepithelium initially evaginates from the embryonic diecenphalon to form a spherical vesicle, the distal part subsequently invaginates resulting in an inner neural retina and an outer pigment epithelium. The in vitro development of the optic cup essentially recapitulates these morphogenetic processes. The forming optic cup structure first evaginates to build a spherical vesicle. Next, the presumptive neural retina flattens, and a so-called hinge region emerges at the border between the neural retina and the future pigment epithelium. Due to an apical constriction at the hinge point, the neural retina finally invaginates leading to a two-layered optic cup consisting of the inner neural retina and the outer pigment epithelium (Eiraku et al., 2012) (Fig. 6B). These data demonstrate that in vitro neural tissue self-organisation can even extent up to complex morphogenetic processes.


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4. Concluding remarks Cultured neural cells exhibit the remarkable ability to spontaneously selforganise into distinct spatial patterns, which can be classified into neural lineage-related primitive architectures (neural rosettes) and complex neural architectures (e.g. cortical neuroepithelium). In particular, data obtained from 3D suspension cultures of PSC-derived NSCs highlight their impressive ability to recapitulate aspects of cortical development in vitro. It will be interesting to see

whether

CNS

histo-architectures

other

than

cortical

or

retinal

neuroepithelia can be efficiently generated from PSCs in the near future. Although generation of neural histo-architectures from stem cells is still in its infancy, it has already become clear that such systems may be useful to study early human neurodevelopmental processes. Beyond the analysis of basic principles governing tissue and organ development, 3D culture systems could also be useful for the investigation of cellular mechanisms underlying neurodevelopmental disorders. Owing to the peculiar set of neural stem and progenitor cells in the developing human brain, animal models might be inadequate for the analysis of human neurodevelopmental defects. Along these lines, patient-specific hiPSCs have been used to study microcephaly in a 3D culture system. Strikingly, patient cells exhibited premature neural differentiation, leading to significantly smaller cerebral organoids in comparison to the ones derived from control cells (Lancaster et al., 2013). This study illustrates that human neurodevelopmental disorders can be analysed in a reductionist human 3D model. Despite these fascinating prospects, the still young field of stem cell-based neural organoids faces a number of technical challenges. For instance, while the initial size of cellular aggregates in the SFEBq system can be controlled using 96-well plates, it appears difficult to avoid their fusion during ongoing cultivation. This inevitably leads to qualitatively different aggregates. Furthermore, embedding



of hPSC-derived neural cells in small matrigel droplets followed by cultivation in a spinning bioreactor system resulted in cerebral organoids with variable content of spatially distinct ‘brain regions’. It will be important to further standardize the 3D culture systems in order to obtain tissue-like structures as homogeneous as possible. This may also include the use of automated cell culture techniques that can be programmed to strictly follow specific culture regimens. Such improvements will be necessary to fully exploit the potential of neural organoids for studying the earliest steps of human nervous system development under physiological and pathophysiological conditions. Acknowledgements We thank Madeline A. Lancaster for kindly providing the picture of the cerebral organoid. We also thank Johannes Jungverdorben and Antonio Martins for providing photomicrographs of 2D and 3D cultures.

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(1051 Words) Figure legends: Figure 1: In vitro rosette formation ability of different neural stem cell populations. (A) The schematic drawing illustrates the different propensity of pre-rosette neural stem cells (pre-rosette NSCs), rosette forming neural stem cells (R-NSCs), long term neuroepithelial stem cells (ltNES) and radial glialike stem cells (NSCFGF2/EGF) to form rosette structures during in vitro culture. These stem cell populations are considered to represent distinct maturation states of neural precursor cells (black arrows). The red color indicates the distribution of apical markers (e.g. N-cadherin, ZO-1, and CD133). While prerosette NSCs already express apical markers, they do not yet show signs of polarisation. Upon propagation in FGF2 pre-rosette cells start forming rosette structures. Concomitantly the apical markers become relocated to the core of the rosette structures generated by both R-NSCs and lt-NES cells. Cell division (green) within R-NSC- and lt-NES-derived rosettes occurs primarily at the rosette core. In contrast, mitoses can be observed in a random fashion in cultures of pre-rosette NSCs and NSCFGF2/EGF. (B) Representative phase contrast images of all four NSC populations generated from hiPSCs. Scale bar: 50 µm. (C) Photomicrographs of hiPSC-derived lt-NES cells stained for the NSC markers PAX6, SOX2, and nestin. The pictures show the arrangement of lt-NES cells in small neural rosettes. Lt-NES cells express the rosette marker promyelocytic leukemia zinc finger protein (PLZF) and the apical marker ZO-1. Note the restriction of ZO-1 immunoreactivity to the core of the rosette. In contrast to R-NSCs, lt-NES cells form small rosettes. Scale bar 25 µm (PAX6 and SOX2), 25 µm (nestin and ZO-1/PLZF). The pictures in


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panel C were kindly provided by Johannes Jungverdorben and are used with his permission. Figure 2: Development of cortical layers. (A) Graphical outline depicting major steps during the development of the human cortex. In the beginning, the cortical neuroepithelium consists of neuroepithelial cells (NECs) that divide at the apical membrane (1). NECs transform into RGCs, which initially give rise to Cajal-Retzius neurons residing in the presumptive layer I (2). Later on they produce intermediate progenitor cells (IPs) and outer radial glia cells (oRGCs) residing in the outer subventricular zone (oSVZ). At that stage IPs and oRGCs significantly contribute to the generation of deep layer neurons (3), followed by upper layer neurons (4). (B) Graphical illustration of cortical progenitors and neurons in a monolayer culture according to Shi et al., 2012b. Two weeks after neural induction of human PSCs, neural tube-like structures contain RGC-like cells that undergo mitosis at the apical side. 25 days after neural induction mitotic pHH3-positive cells can also be observed at more basal regions. This goes along with an emergence of IPs and presumptive oRGCs. With ongoing cultivation deep layer neurons are born first (day 60) followed by upper layer neurons (day 100). However, cortical neurons fail to form distinct neuronal layers. Moreover, their spatial translocation relative to the progenitor cells is unclear. Figure 3: Emergence of primitive cortical layers in suspension culture paradigms.

Photomicrographs

depicting

the

spatial

segregation

of

proliferative and postmitotic cortical cells that have been generated from hiPSCs according to Shi et al., 2012 adapted to suspension culture (unpublished data) (A, B) 50 days after neural induction PAX6-positive neural precursor cells are mainly restricted to rosette structures that display a strong N-CDH-immunoreactivity at their apical side. In contrast, Ki67-positive proliferative cells can also be observed at the outer edge of the neural rosette structure. The distribution of such displaced Ki67-positive cells overlaps with the distribution of TBR2-positive presumptive intermediate progenitors. (C) Postmitotic TBR1-/CTIP2-positive deep-layer neurons are clearly separated from neural rosette structures at that developmental stage. Scale bar: 100 µm



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(A-C). The pictures were kindly provided by Antonio Martins and are used with his permission. Figure 4: HiPSC-derived cerebral organoid. Photomicrograph of a cerebral organoid stained for SOX2 and the early neuronal marker doublecortin (DCX). This cerebral organoid contains a large cortical neuroepithelium surrounding a fluid-filled cavity (arrow). A SOX2-positive choroid plexus-like tissue is present in the middle of the cerebral organoid (asterisk). Scale bar: 500 µm. The photomicrograph was kindly provided by Madeline A. Lancaster and is used with her permission. Figure 5: Emergence of advanced cortical layers in suspension culture paradigms. Schematic drawing depicting the extent of cortical layering observed in the improved SFEBq culture system (Kadoshima) and spinning bioreactor (Lancaster). The denoted days allude to the time points presented in the original publications and are not meant as qualitative comparison. In both culture systems a primitive separation of germinative and neuronal layers can be observed after 42 and 30 days, respectively. Prolonged cultivation leads to an advanced level of stratification. At day 91 (Kadoshima) and day 75 (Lancaster) the germinative layers contain PAX6-positive radial glia cells, TBR2-positive/SOX2-negative presumptive intermediate progenitor cells and TBR2-negative/SOX2-positive presumptive outer radial glia cells. The spatial separation of CTIP2-/TBR1-positive presumptive deep layer and SATB2/BRN2-positive upper layer neurons is compatible with an inside-out pattern of neurogenesis. After 112 days deep layer neurons expressing the mature cortical neuron marker CaMKII-α can be observed in the Kadoshima setting.

Figure

6:

morphogenetic

Self-driven movements

morphogenesis. in

(A)

hESC-derived,

Schematic

showing

self-organised

cortical

neuroepithelia. (1) A cortical neuroepithelium forms at the rim of the cortical cell aggregate; its apical side is oriented outwards. (2, 3) With ongoing cultivation, one end of the neuroepithelium undergoes rounding movements (indicated by dashed arrows), thereby eventually contacting the other, non-


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rolling end. These morphogenetic changes result in the reorientation of the apicobasal cell polarity: the apical side is oriented inwards (towards the forming lumen), while the basal side is oriented outwards. The illustration is partially adapted from Kadoshima et al., 2013 with permission from Mototsugu Eiraku. (B) Graphical illustration showing the in vitro formation of optic cups from mouse and human PSCs. (1) Under appropriate culture conditions a primitive neuroepithelium (grey) starts to evaginate from embryoid body-like aggregates to form a spherical vesicle (dotted arrow). (2, 3) Then, the distal part (i.e. the future neural retina) flattens, and a hinge region (green) emerges at the boarder between the presumptive neural retina (yellow) and pigment epithelium (dark blue). (4) This hinge region undergoes an apical constriction, which leads to the invagination of the neural retina (dotted arrow). Finally, the optic cup consists of an inner, stratified neural retina and an outer pigment epithelium. The illustration is partially adapted from Eiraku et al., 2012 with the permission from Mototsugu Eiraku.



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Production of neural stem cells from human pluripotent stem cells.

Generating kidney tissue from pluripotent stem cells.

Self-Organizing 3D Human Neural Tissue Derived from Induced Pluripotent Stem Cells Recapitulate Alzheimer's Disease Phenotypes.

NPTX1 regulates neural lineage specification from human pluripotent stem cells.

Generating trunk neural crest from human pluripotent stem cells.

Neural retinal regeneration with pluripotent stem cells.

Derivation of Neural Stem Cells from Mouse Induced Pluripotent Stem Cells.

Pluripotent stem cell-derived neural stem cells: From basic research to applications.

Methods for Derivation of Multipotent Neural Crest Cells Derived from Human Pluripotent Stem Cells.

Reversal of cellular phenotypes in neural cells derived from Huntington's disease monkey-induced pluripotent stem cells.

Reprogrammed pluripotent stem cells from somatic cells.

Repression of SIRT1 promotes the differentiation of mouse induced pluripotent stem cells into neural stem cells.

Differentiation of hepatocytes from pluripotent stem cells.

Human intestinal tissue with adult stem cell properties derived from pluripotent stem cells.

A cGMP-applicable expansion method for aggregates of human neural stem and progenitor cells derived from pluripotent stem cells or fetal brain tissue.

Allele-specific knockdown of ALS-associated mutant TDP-43 in neural stem cells derived from induced pluripotent stem cells.

Notch signaling regulates the differentiation of neural crest from human pluripotent stem cells.

Neural differentiation from pluripotent stem cells: The role of natural and synthetic extracellular matrix.

Reconstruction of brain circuitry by neural transplants generated from pluripotent stem cells.

Nephron reconstitution from pluripotent stem cells.

In vitro organogenesis from pluripotent stem cells.

Chemically Induced Reprogramming of Somatic Cells to Pluripotent Stem Cells and Neural Cells.

Induction of early neural precursors and derivation of tripotent neural stem cells from human pluripotent stem cells under xeno-free conditions.

Generation of functional hepatic cells from pluripotent stem cells.