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Heterogeneity in the developmental potential of motor neuron progenitors revealed by clonal analysis of single cells in vitro.

ABSTRACT: BACKGROUND: The differentiation of neural progenitors into distinct classes within the central nervous system occurs over an extended period during which cells become progressively restricted in their fates. In the developing spinal cord, Sonic Hedgehog (Shh) controls neural fates in a concentration-dependent manner by establishing discrete ventral progenitor domains characterized by specific combinations of transcription factors. It is unclear whether motor neuron progenitors can maintain their identities when expanded in vitro and whether their developmental potentials are restricted when exposed to defined extracellular signals. RESULTS: We have generated mice expressing the enhanced green fluorescent protein under the control of the Nkx6.1 promoter, enabling fluorescence-activated cell sorting (FACS), purification and culture of individual spinal progenitors at clonal density, and analysis of their progeny. We demonstrate that cells isolated after progenitor domains are established are heterogeneous with respect to maintaining their identity after in vitro expansion. Most Nkx6.1+ progenitors lose their ventral identity following several divisions in culture, whereas a small subset is able to maintain its identity. Thus, subtype-restricted progenitors from the Nkx6.1+ region are present in the ventral spinal cord, although at a lower frequency than expected. Clones that maintain a motor neuron identity assume a transcriptional profile characteristic of thoracic motor neurons, despite some having been isolated from non-thoracic regions initially. Exposure of progenitors to Bone Morphogenetic Protein-4 induces some dorsal cell type characteristics in their progeny, revealing that lineage-restricted progenitor subtypes are not fully committed to their fates. CONCLUSION: These findings support a model whereby continuous Shh signaling is required to maintain the identity of ventral progenitors isolated from the spinal cord, including motor neuron progenitors, after in vitro expansion. They also demonstrate that pre-patterned neural progenitors isolated from the central nervous system can change their regional identity in vitro to acquire a broader developmental potential.

SUBMITTER: Agalliu D

PROVIDER: S-EPMC2657897 | biostudies-other | 2009

REPOSITORIES: biostudies-other

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Heterogeneity in the developmental potential of motor neuron progenitors revealed by clonal analysis of single cells in vitro.

Agalliu Dritan D Schieren Ira I

Neural development 20090105

<h4>Background</h4>The differentiation of neural progenitors into distinct classes within the central nervous system occurs over an extended period during which cells become progressively restricted in their fates. In the developing spinal cord, Sonic Hedgehog (Shh) controls neural fates in a concentration-dependent manner by establishing discrete ventral progenitor domains characterized by specific combinations of transcription factors. It is unclear whether motor neuron progenitors can maintai ...[more]

PMID: 19123929

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Project description:BackgroundHuman induced pluripotent stem cells (iPSC) have opened new avenues for regenerative medicine. Consequently, iPSC-derived motor neurons have emerged as potentially viable therapies for spinal cord injuries and neurodegenerative disorders including Amyotrophic Lateral Sclerosis. However, direct clinical application of iPSC bears in itself the risk of tumorigenesis and other unforeseeable genetic or epigenetic abnormalities.ResultsEmploying RNA-seq technology, we identified and characterized gene regulatory networks triggered by in vitro chemical reprogramming of iPSC into cells with the molecular features of motor neurons (MNs) whose function in vivo is to innervate effector organs. We present meta-transcriptome signatures of 5 cell types: iPSCs, neural stem cells, motor neuron progenitors, early motor neurons, and mature motor neurons. In strict response to the chemical stimuli, along the MN differentiation axis we observed temporal downregulation of tumor growth factor-? signaling pathway and consistent activation of sonic hedgehog, Wnt/?-catenin, and Notch signaling. Together with gene networks defining neuronal differentiation (neurogenin 2, microtubule-associated protein 2, Pax6, and neuropilin-1), we observed steady accumulation of motor neuron-specific regulatory genes, including Islet-1 and homeobox protein HB9. Interestingly, transcriptome profiling of the differentiation process showed that Ca2+ signaling through cAMP and LPC was downregulated during the conversion of the iPSC to neural stem cells and key regulatory gene activity of the pathway remained inhibited until later stages of motor neuron formation. Pathways shaping the neuronal development and function were well-represented in the early motor neuron cells including, neuroactive ligand-receptor interactions, axon guidance, and the cholinergic synapse formation. A notable hallmark of our in vitro motor neuron maturation in monoculture was the activation of genes encoding G-coupled muscarinic acetylcholine receptors and downregulation of the ionotropic nicotinic acetylcholine receptors expression. We observed the formation of functional neuronal networks as spontaneous oscillations in the extracellular action potentials recorded on multi-electrode array chip after 20?days of differentiation.ConclusionsDetailed transcriptome profile of each developmental step from iPSC to motor neuron driven by chemical induction provides the guidelines to novel therapeutic approaches in the re-construction efforts of muscle innervation.

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Heterogeneity in the developmental potential of motor neuron progenitors revealed by clonal analysis of single cells in vitro.

Publications

Heterogeneity in the developmental potential of motor neuron progenitors revealed by clonal analysis of single cells in vitro.

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