Programming embryonic stem cells to neuronal subtypes
Introduction
One of the tantalizing features of the central nervous system (CNS) is the precision with which nerve cells establish stereotypical neural circuits. Ongoing characterization of the developing CNS is revealing an unprecedented degree of neuronal diversity that corresponds with and likely underlies the richness and specificity of neuronal connectivity and function. Subtype diversification of spinal motor neurons, retinal amacrine cells, cortical layer V pyramidal neurons or olfactory sensory neurons underlies functionally relevant differences in axonal projections, dendritic arborization, and molecular and electrophysiological properties of these neurons [1, 2, 3, 4, 5]. How such neuronal diversity is established during mammalian development remains poorly understood in part due to technical challenges inherent to the analysis of complex and heterogeneous CNS tissue under poorly accessible experimental conditions. Recapitulation of normal neural development with pluripotent embryonic stem cells (ESCs) in vitro might therefore become a powerful and indispensable tool in the field of developmental neurobiology. It provides an unparalleled experimental access to the developing mammalian CNS previously enjoyed only by researchers studying oviparous vertebrates and lower organisms.
Major advances have been reported in directed differentiation of pluripotent cells into distinct classes of nerve cells (reviewed in [6, 7, 8, 9]). These studies firmly established that differentiating pluripotent cells respond correctly to developmental signals that pattern the developing CNS along the rostro-caudal or dorso-ventral axes and give rise to nerve cells expressing congruent region specific markers and neurotransmitter phenotypes. By contrast, less attention has been paid to whether in vitro generated neurons also acquire refined subtype specific properties. This is largely due to the lack of understanding of mechanisms underlying subtype diversification of mammalian nerve cells in vivo. Modeling the acquisition of subtype specific nerve cell identities in a simplified in vitro system might therefore provide a powerful tool to dissect the roles of cell-intrinsic genetic programs, extrinsic diffusible signals, and cell–cell interactions in the fine-grained diversification of developing nerve cells.
Section snippets
Motor neuron subtype diversity
Subtype diversification of neurons is particularly apparent in the motor system. Coherent transmission of motor signals relies on the existence of hundreds of spinal motor neuron subtypes, each communicating with a different muscle group in the periphery (Figure 1a–c). Developmentally encoded motor neuron subtype diversity prescribes the connectivity not only between motor neurons and their muscle targets, but also between motor neuron subtypes and efferent connections from cognate
Specification of subtype identity in ESC-derived motor neurons
Detailed understanding of spinal motor neuron subtype diversity in vivo provides a platform to examine whether similar diversification of motor neurons can be achieved in vitro from differentiating ESCs. Initial methods for differentiation of ESCs to motor neurons relied on two principal patterning signals — RA that induces neuralization and caudalization of ESCs and sonic hedgehog (Hh) that directs ventralization of the spinal neural progenitor cells [18]. ESC-derived motor neurons express bona
Neocortical neuronal subtype diversity
While a strict developmental control of neuronal subtype diversity is a sensible way to ensure reproducible and reliable transmission of signals between the CNS and periphery, central connectivity might benefit from a greater degree of plasticity. Accordingly, specification of neuronal subtype diversity in the developing neocortex does not rely only on developmental programs and local paracrine signals, but also on patterns of innervation by efferent axons [34].
Neocortex develops in the rostral
Specification of principal classes of neocortical neurons from ESCs
Neural tissue induced in the absence of caudalizing signals in the developing embryo acquires rostral forebrain identity [49, 50]. The same principle applies to the patterning of ESC-derived neurons — in the absence of exogenous factors, differentiating ESCs preferentially acquire rostral neural identity [51, 52, 53, 54, 55, 56, 57, 58•, 59]. Neuralization of ESCs under these conditions critically depends on endogenously expressed Fgf5 signal [60] and can be improved by blocking residual BMP,
Regional subtype diversity of in vitro derived cortical pyramidal neurons
Efficient derivation of deep layer cortical nerve cells, many of which exhibit properties of corticofugal layer V pyramidal neurons, raised a question whether in vitro generated pyramidal neurons are largely of one particular subtype identity, a mixture of different identities, or whether they retain plasticity and will acquire their final identities only in response to local cues and efferent innervation as has been demonstrated for primary embryonic cortical tissue.
To examine this question,
Conclusions and future directions
The qualitative leap forward in our ability to differentiate ESCs under conditions that recapitulate normal embryogenesis provides an opportunity to study mammalian neural development in an accessible and convenient system capable of producing virtually unlimited supply of neural cells. This review focused on spinal motor neurons and cortical pyramidal neurons, two classes of nerve cells that were recently demonstrated to acquire refined subtype specific identities in vitro. However, advances
References and recommended reading
Papers of particular interest, published within the period of review, have been highlighted as:
• of special interest
Acknowledgments
Authors of this review acknowledge support by Project A.L.S. grant, NINDS NS058502 and NS055923, and NIH T32 HD055165 Ruth L. Kirschstein National Research Service Award (M.P.). We thank Carolyn Morrison and Fiona Doetsch for critical reading of the manuscript. We apologize to colleagues whose work has not been mentioned due to space limitations.
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