Functional Diversity of ESC-Derived Motor Neuron Subtypes Revealed through Intraspinal Transplantation

Department of Pathology and Cell Biology, Neurology, and Neuroscience, Center for Motor Neuron Biology and Disease, Columbia University Medical Center, New York, NY 10032, USA.
Cell stem cell (Impact Factor: 22.27). 09/2010; 7(3):355-66. DOI: 10.1016/j.stem.2010.07.013
Source: PubMed


Cultured ESCs can form different classes of neurons, but whether these neurons can acquire specialized subtype features typical of neurons in vivo remains unclear. We show here that mouse ESCs can be directed to form highly specific motor neuron subtypes in the absence of added factors, through a differentiation program that relies on endogenous Wnts, FGFs, and Hh-mimicking the normal program of motor neuron subtype differentiation. Molecular markers that characterize motor neuron subtypes anticipate the functional properties of these neurons in vivo: ESC-derived motor neurons grafted isochronically into chick spinal cord settle in appropriate columnar domains and select axonal trajectories with a fidelity that matches that of their in vivo generated counterparts. ESC-derived motor neurons can therefore be programmed in a predictive manner to acquire molecular and functional properties that characterize one of the many dozens of specialized motor neuron subtypes that exist in vivo.

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Available from: Mirza Peljto,
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    • "Specifically, we have demonstrated the ability to generate neuroectoderm with HOX profiles corresponding to specific rhombomeric segments and cervical, thoracic, and lumbosacral vertebral segments. Our findings are distinct from other PSC neural differentiation approaches that unpredictably pattern cells to primarily caudal hindbrain thru cervical HOX profiles (Amoroso et al., 2013; Lee et al., 2007; Li et al., 2005) or to heterogeneous mixtures of cervical, thoracic, and lumbar tissues (Patani et al., 2011; Peljto et al., 2010). Given the data presented in our manuscript, it is perhaps surprising that RA-only patterning approaches can yield HOX6-8 expression, but this may be inherent to the choice of differentiation methods. "
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    ABSTRACT: Colinear HOX expression during hindbrain and spinal cord development diversifies and assigns regional neural phenotypes to discrete rhombomeric and vertebral domains. Despite the precision of HOX patterning in vivo, in vitro approaches for differentiating human pluripotent stem cells (hPSCs) to posterior neural fates coarsely pattern HOX expression thereby generating cultures broadly specified to hindbrain or spinal cord regions. Here, we demonstrate that successive activation of fibroblast growth factor, Wnt/β-catenin, and growth differentiation factor signaling during hPSC differentiation generates stable, homogenous SOX2(+)/Brachyury(+) neuromesoderm that exhibits progressive, full colinear HOX activation over 7 days. Switching to retinoic acid treatment at any point during this process halts colinear HOX activation and transitions the neuromesoderm into SOX2(+)/PAX6(+) neuroectoderm with predictable, discrete HOX gene/protein profiles that can be further differentiated into region-specific cells, e.g., motor neurons. This fully defined approach significantly expands capabilities to derive regional neural phenotypes from diverse hindbrain and spinal cord domains. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.
    Stem Cell Reports 04/2015; 33(4). DOI:10.1016/j.stemcr.2015.02.018 · 5.37 Impact Factor
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    • "MN development can be recapitulated in vitro from mouse or human embryonic stem cells (ESCs), which will form functional spinal MNs under the appropriate culture conditions (Li et al., 2008; Miles et al., 2004; Wichterle et al., 2002). ESC-derivation of MNs depends on the same extrinsic and intrinsic cues that act during normal embryogenesis and has been repeatedly used to investigate subtype-specific developmental pathways in these cells (Jung et al., 2010; Peljto et al., 2010; Soundararajan et al., 2006). We set out to apply this approach to the acquisition of phrenic neuron identity. "
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    ABSTRACT: Air breathing is an essential motor function for vertebrates living on land. The rhythm that drives breathing is generated within the central nervous system and relayed via specialised subsets of spinal motor neurons to muscles that regulate lung volume. In mammals, a key respiratory muscle is the diaphragm, which is innervated by motor neurons in the phrenic nucleus. Remarkably, relatively little is known about how this crucial subtype of motor neuron is generated during embryogenesis. Here, we used direct differentiation of motor neurons from mouse embryonic stem cells as a tool to identify genes that direct phrenic neuron identity. We find that three determinants, Pou3f1, Hoxa5 and Notch, act in combination to promote a phrenic neuron molecular identity. We show that Notch signalling induces Pou3f1 in developing motor neurons in vitro and in vivo. This suggests that the phrenic neuron lineage is established through a local source of Notch ligand at mid-cervical levels. Furthermore, we find that the cadherins Pcdh10, which is regulated by Pou3f1 and Hoxa5, and Cdh10, which is controlled by Pou3f1, are both mediators of like-like clustering of motor neuron cell bodies. This specific Pcdh10/Cdh10 activity might provide the means by which phrenic neurons are assembled into a distinct nucleus. Our study provides a framework for understanding how phrenic neuron identity is conferred and will help to generate this rare and inaccessible yet vital neuronal subtype directly from pluripotent stem cells, thus facilitating subsequent functional investigations.
    Development 02/2014; 141(4):784-94. DOI:10.1242/dev.097188 · 6.46 Impact Factor
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    • "In line with the known role of Pax6 and Emx2 during cortical development, Pax6-positive radial glia (RG) were only capable of acquiring a dorsal but not a ventral phenotype [30]. Additionally, a recent study using ES cell-derived motor neurons generated via a procedure based on intrinsic Wnt, Fgfs and Hh in the absence of retinoic acid, reports that ESNs acquire different motor pool identities [5]. The authors show that molecular markers anticipate functional properties and grafted ESNs settle in appropriate domains, irrespective of neighboring neurons. "
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    ABSTRACT: Enduring reorganization is accepted as a fundamental process of adult neural plasticity. The most dramatic example of this reorganization is the birth and continuously occurring incorporation of new neurons into the pre-existing network of the adult mammalian hippocampus. Based on this phenomenon we transplanted murine embryonic stem (ES)-cell derived neuronal precursors (ESNPs) into murine organotypic hippocampal slice cultures (OHC) and examined their integration. Using a precise quantitative morphological analysis combined with a detailed electrophysiology, we show a region-specific morphological integration of transplanted ESNPs into different subfields of the hippocampal tissue, resulting in pyramidal neuron-like embryonic stem cell-derived neurons (ESNs) in the Cornu Ammonis (CA1 and CA3) and granule neuron-like ESNs in the dentate gyrus (DG), respectively. Subregion specific structural maturation was accompanied by the development of dendritic spines and the generation of excitatory postsynaptic currents (EPSCs). This cell type specific development does not depend upon NMDA-receptor-dependent synaptic transmission. The presented integration approach was further used to determine the cell-autonomous function of the pan-neurotrophin receptor p75 (P75(NTR)), as a possible negative regulator of ESN integration. By this means we used p75(NTR)-deficient ESNPs to study their integration into a WT organotypic environment. We show here that p75(NTR) is not necessary for integration per se but plays a suppressing role in dendritic development.
    PLoS ONE 06/2013; 8(6):e66497. DOI:10.1371/journal.pone.0066497 · 3.23 Impact Factor
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