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GABAergic interneurons are poised with the capacity to shape circuit output via inhibitory gating. How early in the development of medial vestibular nucleus (MVN) are GABAergic neurons recruited for feedforward shaping of outputs to higher centers for spatial navigation? The role of early GABAergic transmission in assembling vestibular circuits for spatial navigation was explored by neonatal perturbation. Immunohistochemistry and confocal imaging were utilized to reveal the expression of parvalbumin (PV)-expressing MVN neurons and their perineuronal nets. Whole-cell patch-clamp recording, coupled with optogenetics, was conducted in vitro to examine the synaptic function of MVN circuitry. Chemogenetic targeting strategy was also employed in vivo to manipulate neuronal activity during navigational tests. We found in rats a neonatal critical period before postnatal day (P) 8 in which competitive antagonization of GABAergic transmission in the MVN retarded maturation of inhibitory neurotransmission, as evidenced by deranged developmental trajectory for excitation/inhibition ratio and an extended period of critical period-like plasticity in GABAergic transmission. Despite increased number of PV-expressing GABAergic interneurons in the MVN, optogenetic-coupled patch-clamp recording indicated null-recruitment of these neurons in tuning outputs along the ascending vestibular pathway. Such perturbation not only offset output dynamics of ascending MVN output neurons, but was further accompanied by impaired vestibular-dependent navigation in adulthood. The same perturbations were however non-consequential when applied after P8. Results highlight neonatal GABAergic transmission as key to establishing feedforward output dynamics to higher brain centers for spatial cognition and navigation.
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Navegación Espacial , Ratas , Animales , Interneuronas , Transmisión Sináptica , Núcleos Vestibulares/metabolismo , Neuronas GABAérgicasRESUMEN
Memory is stored in neural networks via changes in synaptic strength mediated in part by NMDA receptor (NMDAR)-dependent long-term potentiation (LTP). Here we show that a cholecystokinin (CCK)-B receptor (CCKBR) antagonist blocks high-frequency stimulation-induced neocortical LTP, whereas local infusion of CCK induces LTP. CCK-/- mice lacked neocortical LTP and showed deficits in a cue-cue associative learning paradigm; and administration of CCK rescued associative learning deficits. High-frequency stimulation-induced neocortical LTP was completely blocked by either the NMDAR antagonist or the CCKBR antagonist, while application of either NMDA or CCK induced LTP after low-frequency stimulation. In the presence of CCK, LTP was still induced even after blockade of NMDARs. Local application of NMDA induced the release of CCK in the neocortex. These findings suggest that NMDARs control the release of CCK, which enables neocortical LTP and the formation of cue-cue associative memory.
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Colecistoquinina/metabolismo , Potenciación a Largo Plazo/fisiología , Memoria/fisiología , Receptores de N-Metil-D-Aspartato/metabolismo , Animales , Corteza Auditiva/metabolismo , Conducta Animal , Colecistoquinina/genética , Estimulación Eléctrica , Corteza Entorrinal/metabolismo , Femenino , Hipocampo/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , N-Metilaspartato/metabolismo , Neocórtex/metabolismo , Neuronas/metabolismo , Ratas Sprague-Dawley , Receptor de Colecistoquinina B/efectos de los fármacos , Receptor de Colecistoquinina B/metabolismo , Receptores de N-Metil-D-Aspartato/antagonistas & inhibidores , Sinapsis/metabolismoRESUMEN
KEY POINTS: Chemogenetic activation of medial vestibular nucleus-projecting 5-HT neurons resulted in deficits in vestibular-mediated tasks, including negative geotaxis, balance beam and rota-rod tests. The 5-HT1A receptor mediates the vestibular-related behavioural effects of 5-HT in the vestibular nucleus. 5-HT1A receptor activation attenuated evoked excitatory postsynaptic currents and evoked inhibitory postsynaptic currents via a presynaptic mechanism in the vestibular nucleus. ABSTRACT: While the anxiolytic effects of serotonergic neuromodulation are well studied, its role in sensorimotor coordination and postural control is unclear. In this study, we show that an increase of serotonin (5-hydroxytryptamine, 5-HT) at the medial vestibular nucleus (MVN), a brainstem centre for vestibulospinal coordination, by either direct cannula administration or chemogenetic stimulation of MVN-projecting serotonergic neurons, adversely affected performance of rats in vestibular-mediated tasks, including negative geotaxis, balance beam and rota-rod tests. Application of the 5-HT1 and 5-HT7 receptor co-agonist 8-hydroxy-2-(di-n-propylamino) tetralin recapitulated the effect of 5-HT, while co-administration of the specific 5-HT1A receptor antagonist WAY 100135 effectively abolished all 5-HT-induced behavioural deficits. This indicated that 5-HT1A receptors mediated the effects of 5-HT in the rat MVN. Using whole-cell patch-clamp recording, we demonstrated that 5-HT1A receptor activation attenuated both evoked excitatory and evoked inhibitory postsynaptic currents through a presynaptic mechanism in the rat MVN. The results thus highlight the 5-HT1A receptor as the gain controller of vestibular-related brainstem circuits for posture and balance.
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Receptor de Serotonina 5-HT1A , Núcleos Vestibulares , 8-Hidroxi-2-(di-n-propilamino)tetralin , Animales , Potenciales Postsinápticos Excitadores , Ratas , Transmisión SinápticaRESUMEN
The phenotypic instability of adult tissue-derived Schwann cell-like cells (SCLCs) as revealed upon withdrawal of glia-inducing culture supplements limits their clinical utility for cell therapy and disease modelling. We previously overcame this limitation by co-culturing bone marrow-derived SCLCs with neurons purified from developing rat and subsequently human sensory neurons such that direct contact between cell types accomplished the cell-intrinsic switch to the Schwann cell fate. Here, our search for juxtacrine instructive signals found both Notch ligands and neuregulin-1 type III localized on the surface of DRG neurons via live cell immunocytochemistry. Bypassing ligand-induced release of the Notch intracellular domain (NICD) by transient transfection of SCLCs with the pAdlox/V5-His-NICD construct was shown to upregulate ErbB2/3. Interaction of ErbB2/3 with neuregulin-1 type III (NRG1 type III) as presented on neurons then mediated the switch to the Schwann cell fate as demonstrated by expression of S100ß/p75/ Sox10/Krox20. In contrast, treatment of cocultures with γ-secretase inhibitor perturbed Notch signalling in SCLCs and consequently deterred both upregulation of ErbB2/3 and the transition to the Schwann cell fate. Taken together, juxtacrine signalling via Notch is key to the upregulation of ErbB receptors for neuregulin-driven commitment of SCLCs to the Schwann cell fate.
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Médula Ósea , Células de Schwann , Animales , Diferenciación Celular , Células Cultivadas , Técnicas de Cocultivo , Neurregulina-1 , Ratas , Receptor ErbB-2 , Transducción de SeñalRESUMEN
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.
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Células Madre Pluripotentes Inducidas/citología , Modelos Neurológicos , Enfermedades del Sistema Nervioso/fisiopatología , Células-Madre Neurales/patología , Evaluación Preclínica de Medicamentos , Humanos , Células Madre Pluripotentes Inducidas/patología , Enfermedades Neurodegenerativas/fisiopatologíaRESUMEN
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.
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Oído Interno/fisiología , Movimiento , Neuronas/fisiología , Núcleos Vestibulares/fisiología , Animales , Fenómenos Electrofisiológicos , Ratas , Receptores de Superficie Celular/fisiologíaRESUMEN
Disruption of the blood-central nervous system barrier (BCB) is increasingly recognized as a pathological factor in diseases and trauma of the central nervous system. Despite the neuropathological impact, current treatment modalities do not target the BCB; strategies to reconstitute the impaired BCB have been restricted to nutritional and dietary remedies. As an integral cell type in the neurovascular unit, pericytes are crucial to the development, maintenance, and repair of the BCB. As such, pericytes are well poised as cellular agents for reconstitution of the impaired BCB. Here, we summarize recent revelations regarding the role of BCB disruption in diseases and trauma of the central nervous system and highlight how pericytes are harnessed to provide targeted therapeutic effect in each case. This review will also address how recent advances in pericyte derivation strategies can serve to overcome practical hurdles in the clinical use of pericytes.
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Neural progenitor cells (NPCs) derived from human pluripotent stem cells(hPSCs) provide major cell sources for repairing damaged neural circuitry and enabling axonal regeneration after spinal cord injury (SCI). However, the injury niche and inadequate intrinsic factors in the adult spinal cord restrict the therapeutic potential of transplanted NPCs. The Sonic Hedgehog protein (Shh) has crucial roles in neurodevelopment by promoting the formation of motorneurons and oligodendrocytes as well as its recently described neuroprotective features in response to the injury, indicating its essential role in neural homeostasis and tissue repair. In this study, we demonstrate that elevated SHH signaling in hNPCs by inhibiting its negative regulator, SUFU, enhanced cell survival and promoted robust neuronal differentiation with extensive axonal outgrowth, counteracting the harmful effects of the injured niche. Importantly, SUFU inhibition in NPCs exert non-cell autonomous effects on promoting survival and neurogenesis of endogenous cells and modulating the microenvironment by reducing suppressive barriers around lesion sites. The combined beneficial effects of SUFU inhibition in hNPCs resulted in the effective reconstruction of neuronal connectivity with the host and corticospinal regeneration, significantly improving neurobehavioral recovery in recipient animals. These results demonstrate that SUFU inhibition confers hNPCs with potent therapeutic potential to overcome extrinsic and intrinsic barriers in transplantation treatments for SCI.
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The in vitro derivation of Schwann cells from human bone marrow stromal cells (hBMSCs) opens avenues for autologous transplantation to achieve remyelination therapy for post-traumatic neural regeneration. Towards this end, we exploited human induced pluripotent stem-cell-derived sensory neurons to direct Schwann-cell-like cells derived from among the hBMSC-neurosphere cells into lineage-committed Schwann cells (hBMSC-dSCs). These cells were seeded into synthetic conduits for bridging critical gaps in a rat model of sciatic nerve injury. With improvement in gait by 12-week post-bridging, evoked signals were also detectable across the bridged nerve. Confocal microscopy revealed axially aligned axons in association with MBP-positive myelin layers across the bridge in contrast to null in non-seeded controls. Myelinating hBMSC-dSCs within the conduit were positive for both MBP and human nucleus marker HuN. We then implanted hBMSC-dSCs into the contused thoracic cord of rats. By 12-week post-implantation, significant improvement in hindlimb motor function was detectable if chondroitinase ABC was co-delivered to the injured site; such cord segments showed axons myelinated by hBMSC-dSCs. Results support translation into a protocol by which lineage-committed hBMSC-dSCs become available for motor function recovery after traumatic injury to both peripheral and central nervous systems.
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Vaina de Mielina , Células de Schwann , Humanos , Ratas , Animales , Diferenciación Celular , Vaina de Mielina/fisiología , Axones/fisiología , Células Receptoras SensorialesRESUMEN
Vestibular information processed first by the brainstem vestibular nucleus (VN), and further by cerebellum and thalamus, underlies diverse brain function. These include the righting reflexes and spatial cognitive behaviour. While the cerebellar and thalamic circuits that decode vestibular information are known, the importance of VN neurons and the temporal requirements for their maturation that allow developmental consolidation of the aforementioned circuits remains unclear. We show that timely unsilencing of glutamatergic circuits in the VN by NMDA receptor-mediated insertion of AMPAR receptor type 1 (GluA1) subunits is critical for maturation of VN and successful consolidation of higher circuits that process vestibular information. Delayed unsilencing of NMDA receptor-only synapses of neonatal VN neurons permanently decreased their functional connectivity with inferior olive circuits. This was accompanied by delayed pruning of the inferior olive inputs to Purkinje cells and permanent reduction in their plasticity. These derangements led to deficits in associated vestibular righting reflexes and motor co-ordination during voluntary movement. Vestibular-dependent recruitment of thalamic neurons was similarly reduced, resulting in permanently decreased efficiency of spatial navigation. The findings thus show that well-choreographed maturation of the nascent vestibular circuitry is prerequisite for functional integration of vestibular signals into ascending pathways for diverse vestibular-related behaviours.
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Tronco Encefálico , Receptores AMPA , Receptores de N-Metil-D-Aspartato , Núcleos Vestibulares , Humanos , Recién Nacido , Tronco Encefálico/metabolismo , Neuronas/metabolismo , Receptores AMPA/genética , Receptores AMPA/metabolismo , Receptores de N-Metil-D-Aspartato/genética , Receptores de N-Metil-D-Aspartato/metabolismo , Núcleos Vestibulares/metabolismoRESUMEN
Neural stem cells (NSCs) derived from human pluripotent stem cells (hPSCs) are considered a major cell source for reconstructing damaged neural circuitry and enabling axonal regeneration. However, the microenvironment at the site of spinal cord injury (SCI) and inadequate intrinsic factors limit the therapeutic potential of transplanted NSCs. Here, it is shown that half dose of SOX9 in hPSCs-derived NSCs (hNSCs) results in robust neuronal differentiation bias toward motor neuron lineage. The enhanced neurogenic potency is partly attributed to the reduction of glycolysis. These neurogenic and metabolic properties retain after transplantation of hNSCs with reduced SOX9 expression in a contusive SCI rat model without the need for growth factor-enriched matrices. Importantly, the grafts exhibit excellent integration properties, predominantly differentiate into motor neurons, reduce glial scar matrix accumulation to facilitate long-distance axon growth and neuronal connectivity with the host as well as dramatically improve locomotor and somatosensory function in recipient animals. These results demonstrate that hNSCs with half SOX9 gene dosage can overcome extrinsic and intrinsic barriers, representing a powerful therapeutic potential for transplantation treatments for SCI.
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Células-Madre Neurales , Traumatismos de la Médula Espinal , Humanos , Ratas , Animales , Células-Madre Neurales/metabolismo , Traumatismos de la Médula Espinal/genética , Traumatismos de la Médula Espinal/terapia , Traumatismos de la Médula Espinal/metabolismo , Neuronas/metabolismo , Neurogénesis , Cicatrización de Heridas , Factor de Transcripción SOX9/genética , Factor de Transcripción SOX9/metabolismoRESUMEN
Transplantation of oligodendrocyte precursors (OPs) is potentially therapeutic for myelin disorders but a safe and accessible cell source remains to be identified. Here we report a two-step protocol for derivation of highly enriched populations of OPs from bone marrow stromal cells of young adult rats (aMSCs). Neural progenitors among the aMSCs were expanded in non-adherent sphere-forming cultures and subsequently directed along the OP lineage with the use of glial-inducing growth factors. Immunocytochemical and flow cytometric analyses of these cells confirmed OP-like expression of Olig2, PDGFRα, NG2, and Sox10. OPs so derived formed compact myelin both in vitro, as in co-culture with purified neurons, and in vivo, following transplantation into the corpus callosum of neonatal shiverer mice. Not only did the density of myelinated axons in the corpus callosum of recipient shiverer mice reach levels comparable to those in age-matched wild-type mice, but the mean lifespan of recipient shiverer mice also far exceeded those of non-recipient shiverer mice. Our results thus promise progress in harnessing the OP-generating potential of aMSCs towards cell therapy for myelin disorders.
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Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , Células Precursoras de Oligodendrocitos/citología , Células Precursoras de Oligodendrocitos/metabolismo , Animales , Células de la Médula Ósea/citología , Células de la Médula Ósea/metabolismo , Proliferación Celular/fisiología , Criopreservación , Citometría de Flujo , Inmunohistoquímica , Vaina de Mielina/metabolismo , Ratas , Ratas Sprague-Dawley , Remielinización/fisiologíaRESUMEN
Perineuronal nets (PN) restrict neuronal plasticity in the adult brain. We hypothesize that activity-dependent consolidation of PN is required for functional maturation of behavioral circuits. Using the postnatal maturation of brainstem vestibular nucleus (VN) circuits as a model system, we report a neonatal period in which consolidation of central vestibular circuitry for graviception is accompanied by activity-dependent consolidation of chondroitin sulfate (CS)-rich PN around GABAergic neurons in the VN. Postnatal onset of negative geotaxis was used as an indicator for functional maturation of vestibular circuits. Rats display negative geotaxis from postnatal day (P) 9, coinciding with the condensation of CS-rich PN around GABAergic interneurons in the VN. Delaying PN formation, by removal of primordial CS moieties on VN with chondroitinase ABC (ChABC) treatment at P6, postponed emergence of negative geotaxis to P13. Similar postponement was observed following inhibition of GABAergic transmission with bicuculline, in line with the reported role of PN in increasing excitability of parvalbumin neurons. We further reasoned that PN-CS restricts bioavailability of plasticity-inducing factors such as semaphorin 3A (Sema3A) to bring about circuit maturation. Treatment of VN explants with ChABC to liberate PN-bound Sema3A resulted in dendritic growth and arborization, implicating structural plasticity that delays synapse formation. Evidence is thus provided for the role of PN-CS-Sema3A in regulating structural and circuit plasticity at VN interneurons with impacts on the development of graviceptive postural control.
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Matriz Extracelular/metabolismo , Red Nerviosa/metabolismo , Reflejo/fisiología , Semaforina-3A/metabolismo , Vestíbulo del Laberinto/metabolismo , Animales , Condroitina ABC Liasa/farmacología , Proteoglicanos Tipo Condroitín Sulfato/metabolismo , Matriz Extracelular/efectos de los fármacos , Red Nerviosa/efectos de los fármacos , Plasticidad Neuronal/efectos de los fármacos , Plasticidad Neuronal/fisiología , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Parvalbúminas/metabolismo , Ratas , Ratas Sprague-Dawley , Núcleos Vestibulares/efectos de los fármacos , Núcleos Vestibulares/metabolismo , Vestíbulo del Laberinto/efectos de los fármacosRESUMEN
Schwann cell-seeded nerve guidance channels are designed to assist post-traumatic nerve regeneration in the PNS. Chitosan is a natural polymer well suited for tissue engineering as it is biocompatible, non-immunogenic, and biodegradable. Electrospun chitosan nanofibers utilized in nerve guidance channels have the capacity for guiding axonal growth within the channel lumen yet are limited in their capacity to maintain structural integrity within physiological environments. To address this, we attempted genipin crosslinking of chitosan nanofibers. Compared to neat chitosan nanofibers, genipin-treated nanofibers exhibited increased stiffness, resistance to swelling and lysozymal degradation. Furthermore, alignment and proliferation of purified Schwann cell cultures upon genipin-treated substratum was enhanced. When dorsal root ganglion explants were utilized as an in vitro model of peripheral nerve regeneration, emigrating neurons and Schwann cells assumed the uniaxial pattern of aligned electrospun chitosan nanofibers. Neurite growth along the nanofibers led, reaching a frontier more than twice that of the pursuant Schwann cells. Critically, neurite growth rate upon genipin-treated nanofibers demonstrated a 100% increase. Altogether, genipin treatment improves upon the physical and biological properties of chitosan nanofibers towards their utility in nerve guidance channel design.
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Quitosano/farmacología , Iridoides/química , Regeneración Nerviosa/efectos de los fármacos , Neuronas/efectos de los fármacos , Células de Schwann/efectos de los fármacos , Ingeniería de Tejidos/métodos , Animales , Materiales Biocompatibles/química , Materiales Biocompatibles/farmacología , Quitosano/química , Reactivos de Enlaces Cruzados/química , Ganglios Espinales/citología , Ganglios Espinales/efectos de los fármacos , Ganglios Espinales/lesiones , Nanofibras/química , Nanofibras/ultraestructura , Proyección Neuronal/efectos de los fármacos , Neuronas/citología , Cultivo Primario de Células , Ratas , Ratas Sprague-Dawley , Células de Schwann/citología , Nervio Ciático/citología , Nervio Ciático/efectos de los fármacos , Nervio Ciático/lesiones , Técnicas de Cultivo de Tejidos , Andamios del TejidoRESUMEN
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.
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Estimulación Acústica/métodos , Vías Auditivas/efectos de la radiación , Regulación de la Expresión Génica/fisiología , Proteínas Oncogénicas v-fos/metabolismo , Animales , Corteza Auditiva/efectos de los fármacos , Corteza Auditiva/metabolismo , Corteza Auditiva/efectos de la radiación , Vías Auditivas/citología , Vías Auditivas/metabolismo , Bicuculina/farmacología , Mapeo Encefálico , Recuento de Células/métodos , Relación Dosis-Respuesta en la Radiación , Lateralidad Funcional , Antagonistas del GABA/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/efectos de la radiación , Inmunohistoquímica/métodos , Colículos Inferiores/metabolismo , Masculino , Neuronas/metabolismo , Análisis Numérico Asistido por Computador , Ratas , Ratas Sprague-DawleyRESUMEN
This manuscript describes a means to enrich for neural progenitors from the marrow stromal cell (MSC) population and thereafter to direct them to the mature Schwann cell fate. We subjected rat and human MSCs to transient hypoxic conditions (1% oxygen for 16 h) followed by expansion as neurospheres upon low-attachment substratum with epidermal growth factor (EGF)/basic fibroblast growth factor (bFGF) supplementation. Neurospheres were seeded onto poly-D-lysine/laminin-coated tissue culture plastic and cultured in a gliogenic cocktail containing ß-Heregulin, bFGF, and platelet-derived growth factor (PDGF) to generate Schwann cell-like cells (SCLCs). SCLCs were directed to fate commitment via coculture for 2 weeks with purified dorsal root ganglia (DRG) neurons obtained from E14-15 pregnant Sprague Dawley rats. Mature Schwann cells demonstrate persistence in S100ß/p75 expression and can form myelin segments. Cells generated in this manner have potential applications in autologous cell transplantation following spinal cord injury, as well as in disease modeling.
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Células de la Médula Ósea/citología , Diferenciación Celular/fisiología , Células de Schwann/citología , Células Madre/citología , Animales , Células de la Médula Ósea/metabolismo , Hipoxia de la Célula , Células Cultivadas , Técnicas de Cocultivo , Ganglios Espinales/citología , Humanos , Vaina de Mielina/metabolismo , Neurogénesis/fisiología , Neuronas/citología , Ratas , Ratas Sprague-Dawley , Células de Schwann/metabolismo , Células Madre/metabolismoRESUMEN
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.
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Diferenciación Celular , Linaje de la Célula , Células Madre Pluripotentes Inducidas/fisiología , Células-Madre Neurales/fisiología , Remielinización , Células de Schwann/fisiología , Células Receptoras Sensoriales/fisiología , Potenciales de Acción , Animales , Biomarcadores/metabolismo , Línea Celular , Técnicas de Cocultivo , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Pluripotentes Inducidas/trasplante , Red Nerviosa/fisiología , Proteínas del Tejido Nervioso/metabolismo , Células-Madre Neurales/metabolismo , Células-Madre Neurales/trasplante , Enfermedades Neurodegenerativas/metabolismo , Enfermedades Neurodegenerativas/patología , Enfermedades Neurodegenerativas/fisiopatología , Enfermedades Neurodegenerativas/cirugía , Fenotipo , Ratas , Células de Schwann/metabolismo , Células de Schwann/trasplante , Células Receptoras Sensoriales/metabolismo , Células Receptoras Sensoriales/trasplante , Transducción de Señal , Trasplante de Células Madre/métodosRESUMEN
Our ultimate goal of in vitro derivation of Schwann cells (SCs) from adult bone marrow stromal cells (BMSCs) is such that they may be used autologously to assist post-traumatic nerve regeneration. Existing protocols for derivation of SC-like cells from BMSCs fall short in the stability of the acquired phenotype and the functional capacity to myelinate axons. Our experiments indicated that neuro-ectodermal progenitor cells among the human hBMSCs could be selectively expanded and then induced to differentiate into SC-like cells. Co-culture of the SC-like cells with embryonic dorsal root ganglion neurons facilitated contact-mediated signaling that accomplished the switch to fate-committed SCs. Microarray analysis and in vitro myelination provided evidence that the human BMSC-derived SCs were functionally mature. This was reinforced by repair and myelination phenotypes observable in vivo with the derived SCs seeded into a nerve guide as an implant across a critical gap in a rat model of sciatic nerve injury.
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Diferenciación Celular , Células Madre Mesenquimatosas/citología , Células de Schwann/citología , Axones/metabolismo , Biomarcadores , Células Cultivadas , Ganglios Espinales/citología , Ganglios Espinales/metabolismo , Perfilación de la Expresión Génica , Humanos , Inmunofenotipificación , Células Madre Mesenquimatosas/metabolismo , Vaina de Mielina/genética , Vaina de Mielina/metabolismo , Factores de Crecimiento Nervioso/metabolismo , Células-Madre Neurales/citología , Células-Madre Neurales/metabolismo , Neuritas/metabolismo , Neurogénesis , Neuronas/citología , Neuronas/metabolismo , Fenotipo , Células de Schwann/metabolismoRESUMEN
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 IOß 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.
Asunto(s)
Potenciales Postsinápticos Excitadores , Ácido Glutámico/fisiología , Neuronas/fisiología , Núcleo Olivar/fisiología , Rotación , Canales Semicirculares/fisiología , Núcleos Vestibulares/fisiología , Animales , Maleato de Dizocilpina/administración & dosificación , Antagonistas de Aminoácidos Excitadores/administración & dosificación , Femenino , Masculino , Vías Nerviosas/fisiología , Neuronas/metabolismo , Núcleo Olivar/crecimiento & desarrollo , Núcleo Olivar/metabolismo , Proteínas Proto-Oncogénicas c-fos/metabolismo , Ratas , Ratas Sprague-Dawley , Receptores de N-Metil-D-Aspartato/antagonistas & inhibidores , Formación Reticular/metabolismo , Formación Reticular/fisiología , Canales Semicirculares/crecimiento & desarrollo , Núcleos Vestibulares/crecimiento & desarrollo , Núcleos Vestibulares/metabolismo , Vestíbulo del Laberinto/lesionesRESUMEN
BACKGROUND: 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. METHOD: Adherent cultures of BMSCs (rat/human) were subjected to 1 % oxygen for 24 h and then subcultured as neurospheres with epidermal growth factor (EGF) and basic fibroblast growth factor supplementation. Neurospheres and cell progeny were monitored immunocytochemically for marker expression. To generate Schwann cell-like cells, neurospheres were plated out and exposed to gliogenic medium. The resulting cells were co-cultured with purified dorsal root ganglia (rat) neurons and then tested for commitment to the Schwann cell fate. Fate-committed Schwann cells were subjected to in vitro myelination assay. RESULTS: Transient hypoxic treatment increased the size and number of neurospheres generated from both rat and human BMSCs. This effect was EGF-dependent and attenuated with the EGF receptor inhibitor erlotinib. Hypoxia did not affect the capacity of neurospheres to generate neuron- or glia-like precursors. Human Schwann cell-like cells generated from hypoxia-treated BMSCs demonstrated expression of S100ß /p75 and capacity for myelination in vitro. CONCLUSION: Enhancing the yield of neural progenitor cells with hypoxic preconditioning of BMSCs in vitro but without inherent risks of genetic manipulation provides a platform for upscaling production of neural cell derivatives for clinical application in cell-based therapy.