hypoxic-preconditioning-marrow-derived-progenitor-cells-as-source-for

Hypoxic Preconditioning of Marrow-derived Progenitor Cells As a Source for the Generation of Mature Schwann Cells

This manuscript describes a means to enrich for neural progenitors from the marrow stromal cell (MSC) population and thereafter to direct them to the ...

The University of Hong Kong

This review focuses on recent approaches to unravel the capacity of otolith-related brainstem neurons for coding head orientations. In the first section, the spatiotemporal features of central vestibular neurons in response to natural otolithic stimulation are reviewed. Experiments that reveal convergent inputs from bilateral vestibular end organs bear important implications on the processing of spatiotemporal signals and integration of head orientational signals within central otolith neurons. Another section covers the maturation profile of central otolith neurons in the recognition of spatial information. Postnatal changes in the distribution pattern of neuronal subpopulations that subserve the horizontal and vertical otolith systems are highlighted. Lastly, the expression pattern of glutamate receptor subunits and neurotrophin receptors in otolith-related neurons within the vestibular nuclear complex are reviewed in relation to the potential roles of these receptors in the development of vestibular function.

Spatial coding capacity of central otolith neurons.

To address the hypothesis that reactive astrocytes in the basal ganglia of an animal model of Parkinson's disease serve neurotrophic roles, we studied the expression pattern of neurotrophic factors in the basal ganglia of C57/Bl mice that had been treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) to induce the degeneration of nigral dopamine neurons and parkinsonism. MPTP induced significant neuronal degeneration in the substantia nigra pars compacta as detected with Fluoro-Jade B staining, and this was accompanied by an increase in nestin-expressing astrocytes within the caudate-putamen. The number of nestin-positive reactive astrocytes in the caudate-putamen peaked within 3-5 days following MPTP treatment and then declined progressively toward the basal level by 21 days after treatment. Immunofluorescence and confocal microscopy confirmed coexpression of nestin or Ki-67 (cell proliferation marker) in glial fibrillary acid protein-positive astrocytes in the caudate-putamen. Double immunolabeling further revealed immunoreactivities for nerve growth factor (NGF), neurotrophin-3 (NT3), and glial cell line-derived neurotrophic factor (GDNF) in nestin-positive reactive astrocytes. Semiquantification of data obtained from mice 5 days after MPTP injection indicated that the majority of nestin-expressing cells expressed NGF (92%), NT3 (90%), or GDNF (86%). Our results present novel evidence of neurotrophic features among reactive astrocytes in the dopamine-depleted striatum. These nestin-expressing reactive astrocytes may therefore play neurotrophic roles in neural remodeling of the basal ganglia in Parkinson's disease.

Localization of nerve growth factor, neurotrophin-3, and glial cell line-derived neurotrophic factor in nestin-expressing reactive astrocytes in the caudate-putamen of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated C57/Bl mice.

To investigate the corticofugal modulation of acoustic information ascending through the auditory pathway of the rat, immunohistochemical techniques were used to study the functional expression of Fos protein in neurons. With auditory stimulation at different frequencies, Fos expression in the medial geniculate body (MGB), inferior colliculus (IC), superior olivary complex, and cochlear nucleus was examined, and the extent of Fos expression on the two sides was compared. Strikingly, we found densely Fos-labeled neurons in all divisions of the MGB after both presentation of an auditory stimulus and administration of a gamma-aminobutyric acid type A (GABA(A)) antagonist (bicuculline methobromide; BIM) to the auditory cortex. The location of Fos-labeled neurons in the ventral division (MGv) after acoustic stimulation at different frequencies was in agreement with the known tonotopic organization. That no Fos-labeled neurons were found in the MGv with acoustic stimuli alone suggests that the transmission of ascending thalamocortical information is critically governed by corticofugal modulation. The dorsal (DCIC) and external cortices (ECIC) of the IC ipsilateral to the BIM-injected cortex showed a significantly higher number of Fos-labeled neurons than the contralateral IC. However, no difference in the number of Fos-labeled neurons was found between the central nucleus of the IC on either side, indicating that direct corticofugal modulation occurs only in the ECIC and DCIC. Further investigations are needed to assess the functional implications of the morphological differences observed between the descending corticofugal projections to the thalamus and the IC.

Corticofugal modulation of acoustically induced Fos expression in the rat auditory pathway.

Autologous nerve grafts have been the 'gold standard' for treatment of peripheral nerve defects that exceed the critical gap length. To address issues of limited availability of donor nerves and donor site morbidity, we have fabricated chitosan conduits and seeded them with bone marrow stromal cell (BMSC)-derived Schwann cells as an alternative. The derived Schwann cells used were checked for fate commitment. The conduits were tested for efficacy in bridging the critical gap length of 12 mm in sciatic nerves of adult rats. By three months post-operation, mid-shank circumference, nerve conduction velocity, average regenerated myelin area, and myelinated axon count, in nerves bridged with BMSC-derived Schwann cells were similar to those treated with sciatic nerve-derived Schwann cells (p > 0.05) but significantly higher than those bridged with PBS-filled conduits (p < 0.05). Evidence is thus provided in support of the use of chitosan conduits seeded with BMSC-derived Schwann cells to treat critical defects in peripheral nerves. This provides the basis to pursue BMSC as an autologous source of Schwann cells for transplantation therapy in larger animal species.

The regeneration of transected sciatic nerves of adult rats using chitosan nerve conduits seeded with bone marrow stromal cell-derived Schwann cells.

Using sinusoidal oscillations of linear acceleration along both the horizontal and vertical planes to stimulate otolith organs in the inner ear, we charted the postnatal time at which responsive neurons in the rat inferior olive (IO) first showed Fos expression, an indicator of neuronal recruitment into the otolith circuit. Neurons in subnucleus dorsomedial cell column (DMCC) were activated by vertical stimulation as early as P9 and by horizontal (interaural) stimulation as early as P11. By P13, neurons in the β subnucleus of IO (IOβ) became responsive to horizontal stimulation along the interaural and antero-posterior directions. By P21, neurons in the rostral IOβ became also responsive to vertical stimulation, but those in the caudal IOβ remained responsive only to horizontal stimulation. Nearly all functionally activated neurons in DMCC and IOβ were immunopositive for the NR1 subunit of the NMDA receptor and the GluR2/3 subunit of the AMPA receptor. In situ hybridization studies further indicated abundant mRNA signals of the glutamate receptor subunits by the end of the second postnatal week. This is reinforced by whole-cell patch-clamp data in which glutamate receptor-mediated miniature excitatory postsynaptic currents of rostral IOβ neurons showed postnatal increase in amplitude, reaching the adult level by P14. Further, these neurons exhibited subthreshold oscillations in membrane potential as from P14. Taken together, our results support that ionotropic glutamate receptors in the IO enable postnatal coding of gravity-related information and that the rostral IOβ is the only IO subnucleus that encodes spatial orientations in 3-D.

Maturation profile of inferior olivary neurons expressing ionotropic glutamate receptors in rats: role in coding linear accelerations.

We examined the maturation expression profile of tyrosine kinase B (TrkB) receptor in rat vestibular nuclear neurons that were activated by sinusoidal linear acceleration along the horizontal or vertical axis. The otolithic origin of Fos expression in these neurons was confirmed with labyrinthectomized controls and normal controls, which showed only sporadically scattered Fos-labeled neurons in the vestibular nucleus. In P4-6 test rats, no Fos-labeled neurons were found in the vestibular nucleus, but the medial and spinal vestibular neurons showed weak immunoreactivity for TrkB. The intensity of TrkB immunoreactivity in vestibular nuclear neurons progressively increased in the second postnatal week but remained low in adults. From P7 onward, TrkB-expressing neurons responded to horizontal or vertical otolithic stimulation with Fos expression. The number of Fos-labeled vestibular nuclear neurons expressing TrkB increased with age, from 13-43% in P7 rats to 85-90% in adult rats. Our results therefore suggest that TrkB/neurotrophin signaling plays a dominant role in modulating vestibular nuclear neurons for the coding of gravity-related horizontal head movements and for the regulation of vestibular-related behavior during postnatal development.

Postnatal expression of TrkB receptor in rat vestibular nuclear neurons responsive to horizontal and vertical linear accelerations.

The capability of the central vestibular system in utilizing cues arising from the inner ear determines the ability of animals to acquire the sense of head orientations in the three-dimensional space and to shape postural movements. During development, neurons in the vestibular nucleus (VN) show significant changes in their electrophysiological properties. An age-dependent enhancement of membrane excitability is accompanied by a progressive increase in firing rate and discharge regularity. The coding of horizontal and vertical linear motions also exhibits developmental refinement in VN neurons. Further, modification of cell surface receptors, such as glutamate receptors, of developing VN neurons are well-orchestrated in the course of maturation, thereby regulating synaptic efficacy and spatial coding capacity of these neurons in local circuits. Taken together, these characteristic features of VN neurons contribute to developmental establishment of space-centered coordinates within the brain.

Development of neural correlates of linear motion in the rat vestibular nucleus.

The availability of human stem cells heralds a new era for in vitro cell-based modeling of neurodevelopmental and neurodegenerative diseases. Adding to the excitement is the discovery that somatic cells of patients can be reprogrammed to a pluripotent state from which neural lineage cells that carry the disease genotype can be derived. These in vitro cell-based models of neurological diseases hold promise for monitoring of disease initiation and progression, and for testing of new drug treatments on the patient-derived cells. In this review, we focus on the prospective applications of different stem cell types for disease modeling and drug screening. We also highlight how the availability of patient-specific induced pluripotent stem cells (iPS cells) offers a unique opportunity for studying and modeling human neurodevelopmental and neurodegenerative diseases in vitro and for testing small molecules or other potential therapies for these disorders. Finally, the limitations of this technology from the standpoint of reprogramming efficiency and therapeutic safety are discussed.

Induced pluripotent stem cells and neurological disease models.

The recognition of head orientation in the adult involves multi-level integration of inputs within the central vestibular circuitry. How the different inputs are recruited during postnatal development remains unclear. We hypothesize that glutamatergic transmission at the vestibular nucleus contributes to developmental registration of head orientations along the vestibulo-olivary pathway. To investigate the maturation profile by which head rotational signals are registered in the brainstem, we used sinusoidal rotations on the orthogonal planes of the three pairs of semicircular canals. Fos expression was used as readout of neurons responsive to the rotational stimulus. Neurons in the vestibular nucleus and prepositus hypoglossal nucleus responded to all rotations as early as P4 and reached adult numbers by P21. In the reticular formation and inferior olive, neurons also responded to horizontal rotations as early as P4 but to vertical rotations not until P21 and P25, respectively. Neuronal subpopulations that distinguish between rotations activating the orthogonally oriented vertical canals were identifiable in the medial and spinal vestibular nuclei by P14 and in the inferior olivary subnuclei IOβ and IOK by P25. Neonatal perturbation of glutamate transmission in the vestibular nucleus was sufficient to derange formation of this distribution in the inferior olive. This is the first demonstration that developmental refinement of glutamatergic synapses in the central vestibular circuitry is essential for developmental registration of head rotational signals in the brainstem.

Maturation of glutamatergic transmission in the vestibulo-olivary pathway impacts on the registration of head rotational signals in the brainstem of rats.

Although expression quantitative trait locus, eQTL, serves as an explicit indicator of gene-gene associations, challenges remain to disentangle the mechanisms by which genetic variations alter gene expression. Here we combined eQTL and molecular analyses to identify an association between two seemingly non-associated genes in brain expression data from BXD inbred mice, namely Ptpn21 and Nrg3. Using biotinylated receptor tracking and immunoprecipitation analyses, we determined that PTPN21 de-phosphorylates the upstream receptor tyrosine kinase ErbB4 leading to the up-regulation of its downstream signaling. Conversely, kinase-dead ErbB4 (K751R) or phosphatase-dead PTPN21 (C1108S) mutants impede PTPN21-dependent signaling. Furthermore, PTPN21 also induced Elk-1 activation in embryonic cortical neurons and a novel Elk-1 binding motif was identified in a region located 1919bp upstream of the NRG3 initiation codon. This enables PTPN21 to promote NRG3 expression through Elk-1, which provides a biochemical mechanism for the PTPN21-NRG3 association identified by eQTL. Biologically, PTPN21 positively influences cortical neuronal survival and, similar to Elk-1, it also enhances neuritic length. Our combined approaches show for the first time, a link between NRG3 and PTPN21 within a signaling cascade. This may explain why these two seemingly unrelated genes have previously been identified as risk genes for schizophrenia.

PTPN21 exerts pro-neuronal survival and neuritic elongation via ErbB4/NRG3 signaling.

: Strategies that exploit induced pluripotent stem cells (iPSCs) to derive neurons have relied on cocktails of cytokines and growth factors to bias cell-signaling events in the course of fate choice. These are often costly and inefficient, involving multiple steps. In this study, we took an alternative approach and selected 5 small-molecule inhibitors of key signaling pathways in an 8-day program to induce differentiation of human iPSCs into sensory neurons, reaching ≥80% yield in terms of marker proteins. Continuing culture in maintenance medium resulted in neuronal networks immunopositive for synaptic vesicle markers and vesicular glutamate transporters suggestive of excitatory neurotransmission. Subpopulations of the derived neurons were electrically excitable, showing tetrodotoxin-sensitive action potentials in patch-clamp experiments. Coculture of the derived neurons with rat Schwann cells under myelinating conditions resulted in upregulated levels of neuronal neuregulin 1 type III in conjunction with the phosphorylated receptors ErbB2 and ErbB3, consistent with amenability of the neuritic network to myelination. As surrogates of embryonic dorsal root ganglia neurons, the derived sensory neurons provided contact-dependent cues to commit bone marrow-derived Schwann cell-like cells to the Schwann cell fate. Our rapid and efficient induction protocol promises not only controlled differentiation of human iPSCs into sensory neurons, but also utility in the translation to a protocol whereby human bone marrow-derived Schwann cells become available for autologous transplantation and remyelination therapy.

Human Induced Pluripotent Cell-Derived Sensory Neurons for Fate Commitment of Bone Marrow-Derived Schwann Cells: Implications for Remyelination Therapy.

Bone marrow stromal cells (BMSCs) are attractive as a source of neural progenitors for ex vivo generation of neurons and glia. Limited numbers of this subpopulation, however, hinder translation into autologous cell-based therapy. Here, we demonstrate rapid and efficient conditioning with hypoxia to enrich for these neural progenitor cells prior to further expansion in neurosphere culture.

Rapid and efficient generation of neural progenitors from adult bone marrow stromal cells by hypoxic preconditioning.

Strategies that exploit induced pluripotent stem cells (iPSCs) to derive neurons have relied on cocktails of cytokines and growth factors to bias cell-signaling events in the course of fate choice. These are often costly and inefficient, involving multiple steps. In this study, we took an alternative approach and selected 5 small-molecule inhibitors of key signaling pathways in an 8-day program to induce differentiation of human iPSCs into sensory neurons, reaching ≥80% yield in terms of marker proteins. Continuing culture in maintenance medium resulted in neuronal networks immunopositive for synaptic vesicle markers and vesicular glutamate transporters suggestive of excitatory neurotransmission. Subpopulations of the derived neurons were electrically excitable, showing tetrodotoxin-sensitive action potentials in patch-clamp experiments. Coculture of the derived neurons with rat Schwann cells under myelinating conditions resulted in upregulated levels of neuronal neuregulin 1 type III in conjunction with the phosphorylated receptors ErbB2 and ErbB3, consistent with amenability of the neuritic network to myelination. As surrogates of embryonic dorsal root ganglia neurons, the derived sensory neurons provided contact-dependent cues to commit bone marrow-derived Schwann cell-like cells to the Schwann cell fate. Our rapid and efficient induction protocol promises not only controlled differentiation of human iPSCs into sensory neurons, but also utility in the translation to a protocol whereby human bone marrow-derived Schwann cells become available for autologous transplantation and remyelination therapy. Stem Cells Translational Medicine 2017;6:369-381.

Article	Year
Spatial coding capacity of central otolith neurons. Experimental brain research\| PubMed ID: 16683136	2006
Localization of nerve growth factor, neurotrophin-3, and glial cell line-derived neurotrophic factor in nestin-expressing reactive astrocytes in the caudate-putamen of 1-methyl-4-phenyl-1,2,... The Journal of comparative neurology\| PubMed ID: 16802332	2006
Corticofugal modulation of acoustically induced Fos expression in the rat auditory pathway. The Journal of comparative neurology\| PubMed ID: 17278128	2007
The regeneration of transected sciatic nerves of adult rats using chitosan nerve conduits seeded with bone marrow stromal cell-derived Schwann cells. Biomaterials\| PubMed ID: 20950852	2011
Maturation profile of inferior olivary neurons expressing ionotropic glutamate receptors in rats: role in coding linear accelerations. Brain structure & function\| PubMed ID: 22706760	2013
Postnatal expression of TrkB receptor in rat vestibular nuclear neurons responsive to horizontal and vertical linear accelerations. The Journal of comparative neurology\| PubMed ID: 22806574	2013
Development of neural correlates of linear motion in the rat vestibular nucleus. Sheng li xue bao : [Acta physiologica Sinica]\| PubMed ID: 24553868	2014
Induced pluripotent stem cells and neurological disease models. Sheng li xue bao : [Acta physiologica Sinica]\| PubMed ID: 24553870	2014
Maturation of glutamatergic transmission in the vestibulo-olivary pathway impacts on the registration of head rotational signals in the brainstem of rats. Brain structure & function\| PubMed ID: 25304399	2016
PTPN21 exerts pro-neuronal survival and neuritic elongation via ErbB4/NRG3 signaling. The international journal of biochemistry & cell biology\| PubMed ID: 25681686	2015
Human Induced Pluripotent Cell-Derived Sensory Neurons for Fate Commitment of Bone Marrow-Derived Schwann Cells: Implications for Remyelination Therapy. Stem cells translational medicine\| PubMed ID: 27629765	2016
Rapid and efficient generation of neural progenitors from adult bone marrow stromal cells by hypoxic preconditioning. Stem cell research & therapy\| PubMed ID: 27717376	2016
Human Induced Pluripotent Cell-Derived Sensory Neurons for Fate Commitment of Bone Marrow-Derived Schwann Cells: Implications for Remyelination Therapy. Stem cells translational medicine\| PubMed ID: 28191772	2017

Daisy Kwok-Yan Shum

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