RESUMEN
Olfactory ensheathing cells (OECs) are unique glial cells found in both the central and peripheral nervous systems where they support the continuous axonal outgrowth of immature olfactory sensory neurons to their targets. Here we show that following severe spinal cord injury, olfactory bulb-derived OECs transplanted near the injury site modify the normally inhibitory glial scar and facilitate axon regeneration past the scar border and into the lesion center. To understand the mechanisms underlying the reparative properties of such transplanted OECs, we used single-cell RNA-sequencing to study their gene expression programs. Our analyses revealed five diverse subtypes of OECs, each expressing novel marker genes and pathways indicative of progenitor, axonal regeneration and repair, secreted molecules, or microglia-like functions. As expected, we found substantial overlap of OEC genes with those of Schwann cells, but also with astrocytes, oligodendrocytes and microglia. We confirmed established markers on cultured OECs, and then localized select top genes of OEC subtypes in rat olfactory bulb tissue. In addition, we present evidence that OECs secrete both Reelin and Connective tissue growth factor, extracellular matrix molecules which are important for neural repair and axonal outgrowth. Our results support that adult OECs are a unique hybrid glia, some with progenitor characteristics, and that their gene expression patterns indicate diverse functions related to wound healing, injury repair and axonal regeneration.
RESUMEN
Having observed that electrical spinal cord stimulation and training enabled four patients with paraplegia with motor complete paralysis to regain voluntary leg movement, the underlying mechanisms involved in forming the newly established supraspinal-spinal functional connectivity have become of great interest. van den Brand et al. (Science 336: 1182-1185, 2012) subsequently, demonstrated the recovery, in response to spinal electro-neuromodulation and locomotor training, of voluntary stepping of the lower limbs in rats that received a lesion that is assumed to eliminate all long-descending cortical axons that project to lumbosacral segments. Here, we used a similar spinal lesion in rats to eliminate long-descending axons to determine whether a novel, trained motor behavior triggered by a unique auditory cue learned before a spinal lesion, could recover after the lesion. Hindlimb stepping recovered 1 mo after the spinal injury, but only after 2 mo, the novel and unique audio-triggered behavior was recovered, meaning that not only was a novel connectivity formed but also further evidence suggested that this highly unique behavioral response was independent of the recovery of the circuitry that generated stepping. The unique features of the newly formed supraspinal-spinal connections that mediated the recovery of the trained behavior is consistent with a guidance mechanism(s) that are highly use dependent.NEW & NOTEWORTHY Electrical spinal cord stimulation has enabled patients with paraplegia to regain voluntary leg movement, and so the underlying mechanisms involved in this recovery are of great interest. Here, we demonstrate in rodents the recovery of trained motor behavior after a spinal lesion. Rodents were trained to kick their right hindlimb in response to an auditory cue. This behavior recovered 2 mo after the paralyzing spinal cord injury but only with the assistance of electrical spinal cord stimulation.
Asunto(s)
Aprendizaje , Paraplejía/fisiopatología , Estimulación de la Médula Espinal/métodos , Médula Espinal/fisiopatología , Animales , Axones/fisiología , Encéfalo/fisiopatología , Potenciales Evocados Motores , Miembro Posterior/inervación , Miembro Posterior/fisiopatología , Neuronas Motoras/fisiología , Movimiento , Paraplejía/terapia , Ratas , Ratas Sprague-DawleyRESUMEN
Mice missing either Reelin or Disabled-1 (Dab1) exhibit dorsal horn neuronal positioning errors and display heat hypersensitivity and mechanical insensitivity. Reelin binds its receptors, apolipoprotein E receptor 2 and very low-density lipoprotein receptor, leading to the recruitment and phosphorylation of Dab1 and activation of downstream pathways that regulate neuronal migration. Previously, we reported that 70% of Dab1 laminae I-II neurons co-expressed LIM-homeobox transcription factor 1-beta (Lmx1b). Here, we asked whether Reelin-expressing dorsal horn neurons co-express Lmx1b, are mispositioned in dab1 mutants, and contribute to nociceptive abnormalities. About 90% of Reelin-labeled neurons are Lmx1b-positive in laminae I-II, confirming that most Reelin and Dab1 neurons are glutamatergic. We determined that Reelin-Lmx1b and Dab1-Lmx1b dorsal horn neurons are separate populations, and together, comprise 37% of Lmx1b-positive cells within and above the Isolectin B4 (IB4) layer in wild-type mice. Compared to wild-type mice, dab1 mutants have a reduced area of laminae I-II outer (above the IB4 layer), more Reelin-Lmx1b neurons within the IB4 layer, and fewer Reelin-Lmx1b neurons within the lateral reticulated area of lamina V and lateral spinal nucleus. Interestingly, both Reelin- and Dab1-labeled dorsal horn neurons sustain similar positioning errors in mutant mice. After noxious thermal and mechanical stimulation, Reelin, Lmx1b, and Reelin-Lmx1b neurons expressed Fos in laminae I-II and the lateral reticulated area in wild-type mice and, therefore, participate in nociceptive circuits. Together, our data suggest that disruption of the Reelin-signaling pathway results in neuroanatomical abnormalities that contribute to the nociceptive changes that characterize these mutant mice.
Asunto(s)
Moléculas de Adhesión Celular Neuronal , Proteínas de la Matriz Extracelular , Animales , Moléculas de Adhesión Celular Neuronal/genética , Movimiento Celular , Proteínas de la Matriz Extracelular/genética , Ratones , Proteínas del Tejido Nervioso/genética , Neuronas , Células del Asta Posterior , Proteína Reelina , Serina Endopeptidasas , Transducción de Señal , Asta Dorsal de la Médula EspinalRESUMEN
Reelin (Reln) and Disabled-1 (Dab1) participate in the Reln-signaling pathway and when either is deleted, mutant mice have the same spinally mediated behavioral abnormalities, increased sensitivity to noxious heat and a profound loss in mechanical sensitivity. Both Reln and Dab1 are highly expressed in dorsal horn areas that receive and convey nociceptive information, Laminae I-II, lateral Lamina V, and the lateral spinal nucleus (LSN). Lamina I contains both projection neurons and interneurons that express Neurokinin-1 receptors (NK1Rs) and they transmit information about noxious heat both within the dorsal horn and to the brain. Here, we ask whether the increased heat nociception in Reln and dab1 mutants is due to incorrectly positioned dorsal horn neurons that express NK1Rs. We found more NK1R-expressing neurons in Reln-/- and dab1-/- Laminae I-II than in their respective wild-type mice, and some NK1R neurons co-expressed Dab1 and the transcription factor Lmx1b, confirming their excitatory phenotype. Importantly, heat stimulation in dab1-/- mice induced Fos in incorrectly positioned NK1R neurons in Laminae I-II. Next, we asked whether these ectopically placed and noxious-heat responsive NK1R neurons participated in pain behavior. Ablation of the superficial NK1Rs with an intrathecal injection of a substance P analog conjugated to the toxin saporin (SSP-SAP) eliminated the thermal hypersensitivity of dab1-/- mice, without altering their mechanical insensitivity. These results suggest that ectopically positioned NK1R-expressing neurons underlie the heat hyperalgesia of Reelin-signaling pathway mutants, but do not contribute to their profound mechanical insensitivity.
Asunto(s)
Moléculas de Adhesión Celular Neuronal/fisiología , Proteínas de la Matriz Extracelular/fisiología , Hiperalgesia/fisiopatología , Proteínas del Tejido Nervioso/fisiología , Células del Asta Posterior/fisiología , Receptores de Neuroquinina-1/fisiología , Serina Endopeptidasas/fisiología , Animales , Moléculas de Adhesión Celular Neuronal/genética , Proteínas de la Matriz Extracelular/genética , Calor , Masculino , Ratones Noqueados , Proteínas del Tejido Nervioso/genética , Células del Asta Posterior/metabolismo , Receptores de Neuroquinina-1/metabolismo , Proteína Reelina , Serina Endopeptidasas/genética , Transducción de Señal , Médula Espinal/fisiopatologíaRESUMEN
Olfactory ensheathing cells (OECs) are unique glia that support axon outgrowth in the olfactory system, and when used as cellular therapy after spinal cord injury, improve recovery and axon regeneration. Here we assessed the effects of combining OEC transplantation with another promising therapy, epidural electrical stimulation during a rehabilitative motor task. Sprague-Dawley rats received a mid-thoracic transection and transplantation of OECs or fibroblasts (FBs) followed by lumbar stimulation while climbing an inclined grid. We injected pseudorabies virus (PRV) into hindlimb muscles 7â¯months post-injury to assess connectivity across the transection. Analyses showed that the number of serotonergic (5-HT) axons that crossed the rostral scar border and the area of neurofilament-positive axons in the injury site were both greater in OEC- than FB-treated rats. We detected PRV-labeled cells rostral to the transection and remarkable evidence of 5-HT and PRV axons crossing the injury site in 1 OEC- and 1 FB-treated rat. The axons that crossed suggested either axon regeneration (OEC) or small areas of probable tissue sparing (FB). Most PRV-labeled thoracic neurons were detected in laminae VII or X, and ~25% expressed Chx10, a marker for V2a interneurons. These findings suggest potential regeneration or sparing of circuits that connect thoracic interneurons to lumbar somatic motor neurons. Despite evidence of axonal connectivity, no behavioral changes were detected in this small-scale study. Together these data suggest that when supplemented with epidural stimulation and climbing, OEC transplantation can increase axonal growth across the injury site and may promote recovery of propriospinal circuitry.
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Axones/fisiología , Trasplante de Células/métodos , Terapia por Estimulación Eléctrica/métodos , Neuroglía/fisiología , Bulbo Olfatorio/citología , Traumatismos de la Médula Espinal/patología , Traumatismos de la Médula Espinal/terapia , Animales , Modelos Animales de Enfermedad , Espacio Epidural/fisiología , Femenino , Proteína Ácida Fibrilar de la Glía/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Neuroglía/trasplante , Ratas , Ratas Sprague-Dawley , Serotonina/metabolismo , Transducción GenéticaRESUMEN
The Reelin-signaling pathway is essential for correct neuronal positioning within the central nervous system. Mutant mice with a deletion of Reelin, its lipoprotein receptors, or its intracellular adaptor protein Disabled-1 (Dab1), exhibit nociceptive abnormalities: thermal (heat) hyperalgesia and reduced mechanical sensitivity. To determine dorsal horn alterations associated with these nociceptive abnormalities, we first characterized the correctly positioned Dab1 neurons in wild-type and mispositioned neurons in Reelin-signaling pathway mutant lumbar spinal cord. Using immunofluorescence, we found that 70% of the numerous Dab1 neurons in Reln+/+ laminae I-II and 67% of those in the lateral reticulated area and lateral spinal nucleus (LSN) co-express the LIM-homeobox transcription factor 1 beta (Lmx1b), an excitatory glutamatergic neuron marker. Evidence of Dab1- and Dab1-Lmx1b neuronal positioning errors was found within the isolectin B4 terminal region of Reln-/- lamina IIinner and in the lateral reticulated area and LSN, where about 50% of the Dab1-Lmx1b neurons are missing. Importantly, Dab1-Lmx1b neurons in laminae I-II and the lateral reticulated area express Fos after noxious thermal or mechanical stimulation and thus participate in these circuits. In another pain relevant locus - the lateral cervical nucleus (LCN), we also found about a 50% loss of Dab1-Lmx1b neurons in Reln-/- mice. We suggest that extensively mispositioned Dab1 projection neurons in the lateral reticulated area, LSN, and LCN and the more subtle positioning errors of Dab1 interneurons in laminae I-II contribute to the abnormalities in pain responses found in Reelin-signaling pathway mutants.
Asunto(s)
Proteínas con Homeodominio LIM/genética , Proteínas del Tejido Nervioso/genética , Nocicepción , Células del Asta Posterior/metabolismo , Factores de Transcripción/genética , Animales , Moléculas de Adhesión Celular Neuronal/genética , Moléculas de Adhesión Celular Neuronal/metabolismo , Proteínas de la Matriz Extracelular/genética , Proteínas de la Matriz Extracelular/metabolismo , Proteínas con Homeodominio LIM/metabolismo , Ratones , Proteínas del Tejido Nervioso/metabolismo , Células del Asta Posterior/fisiología , Proteína Reelina , Serina Endopeptidasas/genética , Serina Endopeptidasas/metabolismo , Factores de Transcripción/metabolismoRESUMEN
Genetic diseases occur in breeds used for law enforcement. As important team members, dogs are expected to operate at peak performance for several years and are significant investments for both the initial purchase and extensive, specialized training. Previous studies have not focused on causes for retirement or euthanasia as genetic (inherited) versus acquired (environmental). We performed direct mutational analysis for breed-specific conditions on samples from 304 dogs including 267 law enforcement (122 US, 87 Israeli, and 58 Polish) and 37 search and rescue dogs. Genetic testing identified 29% (n = 89) of the dogs tested to be carriers of a genetic mutation and 6% (n = 19) to be at risk for a debilitating inherited condition that may eventually impair the dog's ability to work. At-risk dogs included Labrador Retrievers (n = 4) with exercise-induced collapse, Bloodhounds (n = 2) with degenerative myelopathy (DM), and German Shepherd dogs with DM (n = 12) or leukocyte adhesion deficiency, type III (n = 1). A substantial number of working dogs were shown to be at risk for genetic conditions that may shorten the dog's career. The loss of dogs, due to early retirement or euthanasia, as a result of preventable genetic conditions has an emotional cost to handlers and financial cost to service organizations that can be avoided with genetic screening prior to breeding, buying, or training.
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Enfermedades de los Perros/epidemiología , Perros/genética , Enfermedades Genéticas Congénitas/veterinaria , Animales , Cruzamiento , Enfermedades de los Perros/genética , Tamización de Portadores Genéticos , Enfermedades Genéticas Congénitas/epidemiología , Enfermedades Genéticas Congénitas/genética , Predisposición Genética a la Enfermedad , Genotipo , Encuestas Epidemiológicas , Israel/epidemiología , Polonia/epidemiología , Especificidad de la Especie , Estados Unidos/epidemiologíaRESUMEN
Certain racial and ethnic groups, individuals with disabilities, and those from low socioeconomic backgrounds remain underrepresented (UR) in the biomedical sciences. This underrepresentation becomes more extreme at each higher education stage. To support UR scholars during the critical transition from baccalaureate to PhD, we established an intensive, 1-yr postbaccalaureate training program. We hypothesized that this intervention would strengthen each participant's competitiveness for leading PhD programs and build a foundation of skills and self-efficacy important for success during and after graduate school. Scholar critical analysis skills, lab technique knowledge, and Graduate Record Examination scores all improved significantly during the program. Scholars reported significant confidence growth in 21 of 24 categories related to success in research careers. In 5 yr, 91% (41/45) of scholars transitioned directly into PhD programs. Importantly, 40% (18/45) of participating postbaccalaureate scholars had previously been declined acceptance into graduate school; however, 17/18 of these scholars directly entered competitive PhD programs following our training program. Alumni reported they were "extremely well" prepared for graduate school, and 95% (39/41) are currently making progress to graduation with a PhD. In conclusion, we report a model for postbaccalaureate training that could be replicated to increase participation and success among UR scholars in the biomedical sciences.
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Investigación Biomédica/educación , Educación de Postgrado , Femenino , Humanos , Masculino , Grupos Minoritarios/educación , Evaluación de Programas y Proyectos de SaludRESUMEN
UNLABELLED: Multiple neural and peripheral cell types rapidly respond to tissue damage after spinal cord injury to form a structurally and chemically inhibitory scar that limits axon regeneration. Astrocytes form an astroglial scar and produce chondroitin sulfate proteoglycans (CSPGs), activate microglia, and recruit blood-derived immune cells to the lesion for debris removal. One beneficial therapy, olfactory ensheathing cell (OEC) transplantation, results in functional improvements and promotes axon regeneration after spinal cord injury. The lack of an OEC-specific marker, however, has limited the investigation of mechanisms underlying their proregenerative effects. We compared the effects of enhanced green fluorescent protein-labeled fibroblast (FB) and OEC transplants acutely after a complete low-thoracic spinal cord transection in adult rats. We assessed the preservation of neurons and serotonergic axons, the levels of inhibitory CSPGs and myelin debris, and the extent of immune cell activation between 1 and 8 weeks postinjury. Our findings indicate that OECs survive longer than FBs post-transplantation, preserve axons and neurons, and reduce inhibitory molecules in the lesion core. Additionally, we show that OECs limit immune-cell activation and infiltration, whereas FBs alter astroglial scar formation and increase immune-cell infiltration and concomitant secondary tissue damage. Administration of cyclosporine-A to enhance graft survival demonstrated that immune suppression can augment OEC contact-mediated protection of axons and neurons during the first 2 weeks postinjury. Collectively, these data suggest that OECs have neuroprotective and immunomodulatory mechanisms that create a supportive environment for neuronal survival and axon regeneration after spinal cord injury. SIGNIFICANCE STATEMENT: Spinal cord injury creates physical and chemical barriers to axon regeneration. We used a complete spinal cord transection model and olfactory ensheathing cell (OEC) or fibroblast (FB; control) transplantation as a repair strategy. OECs, but not FBs, intermingled with astrocytes, facilitated astroglial scar border formation and sequestered invading peripheral cells. OECs attenuated immune cell infiltration, reduced secondary tissue damage, protected neurons and axons in the lesion core, and helped clear myelin debris. Immunosuppression enhanced survival of OECs and FBs, but only OEC transplantation promoted scaffold formation in the lesion site that facilitated axon regeneration and neuron preservation.
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Trasplante de Células/métodos , Regeneración Nerviosa/fisiología , Bulbo Olfatorio/citología , Neuronas Receptoras Olfatorias/fisiología , Traumatismos de la Médula Espinal/cirugía , Animales , Axones/efectos de los fármacos , Axones/fisiología , Células Cultivadas , Corteza Cerebral/patología , Ciclosporinas/farmacología , Ciclosporinas/uso terapéutico , Modelos Animales de Enfermedad , Fibroblastos/fisiología , Fibroblastos/trasplante , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/fisiología , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Vaina de Mielina/patología , Regeneración Nerviosa/efectos de los fármacos , Proteínas del Tejido Nervioso/metabolismo , Infiltración Neutrófila/fisiología , Neuronas Receptoras Olfatorias/trasplante , Ratas , Ratas Sprague-Dawley , Ratas Transgénicas , Serotonina/metabolismo , Traumatismos de la Médula Espinal/tratamiento farmacológico , Traumatismos de la Médula Espinal/fisiopatologíaRESUMEN
The unique glia located in the olfactory system, called olfactory ensheathing cells (OECs), are implicated as an attractive choice for transplantation therapy following spinal cord injury because of their pro-regenerative characteristics. Adult OECs are thought to improve functional recovery and regeneration after injury by secreting neurotrophic factors and making cell-to-cell contacts with regenerating processes, but the mechanisms are not well understood. We show first that α7 integrin, a laminin receptor, is highly expressed at the protein level by OECs throughout the olfactory system, i.e., in the olfactory mucosa, olfactory nerve, and olfactory nerve layer of the olfactory bulb. Then we asked if OECs use the α7 integrin receptor directly to promote neurite outgrowth on permissive and neutral substrates, in vitro. We co-cultured α7+/+ and α7lacZ/lacZ postnatal cerebral cortical neurons with α7+/+ or α7lacZ/lacZ OECs and found that genotype did not effect the ability of OECs to enhance neurite outgrowth by direct contact. Loss of α7 integrin did however significantly decrease the motility of adult OECs in transwell experiments. Twice as many α7+/+ OECs migrated through laminin-coated transwells compared to α7+/+ OECs on poly-L-lysine (PLL). This is in contrast to α7lacZ/lacZ OECs, which showed no migratory preference for laminin substrate over PLL. These results demonstrate that OECs express α7 integrin, and that laminin and its α7 integrin receptor contribute to adult OEC migration in vitro and perhaps also in vivo.
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Antígenos CD/metabolismo , Cadenas alfa de Integrinas/metabolismo , Laminina/metabolismo , Animales , Movimiento Celular , Células Cultivadas , Técnicas de Cocultivo , Distroglicanos/metabolismo , Inmunohistoquímica , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Microscopía Confocal , Neuritas/fisiología , Bulbo Olfatorio/citología , Bulbo Olfatorio/metabolismo , Proteína Marcadora Olfativa/metabolismo , Neuronas Receptoras Olfatorias/metabolismoRESUMEN
The regenerative capacity of adult CNS neurons after injury is strongly inhibited by the spinal cord lesion site environment that is composed primarily of the reactive astroglial scar and invading meningeal fibroblasts. Olfactory ensheathing cell (OEC) transplantation facilitates neuronal survival and functional recovery after a complete spinal cord transection, yet the mechanisms by which this recovery occurs remain unclear. We used a unique multicellular scar-like culture model to test if OECs promote neurite outgrowth in growth-inhibitory areas. Astrocytes were mechanically injured and challenged by meningeal fibroblasts to produce key inhibitory elements of a spinal cord lesion. Neurite outgrowth of postnatal cerebral cortical neurons was assessed on three substrates: quiescent astrocyte control cultures, reactive astrocyte scar-like cultures, and scar-like cultures with OECs. Initial results showed that OECs enhanced total neurite outgrowth of cortical neurons in a scar-like environment by 60%. We then asked if the neurite growth-promoting properties of OECs depended on direct alignment between neuronal and OEC processes. Neurites that aligned with OECs were nearly three times longer when they grew on inhibitory meningeal fibroblast areas and twice as long on reactive astrocyte zones compared to neurites not associated with OECs. Our results show that OECs can independently enhance neurite elongation and that direct OEC-neurite cell contact can provide a permissive substrate that overcomes the inhibitory nature of the reactive astrocyte scar border and the fibroblast-rich spinal cord lesion core.
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Regeneración Nerviosa/fisiología , Neuritas/patología , Neuronas/citología , Bulbo Olfatorio/citología , Animales , Astrocitos/patología , Células Cultivadas , Corteza Cerebral/patología , Cicatriz/patología , Técnicas de Cocultivo , Neuritas/fisiología , Neurogénesis , Ratas , Traumatismos de la Médula Espinal/fisiopatologíaRESUMEN
Components of the Reelin-signaling pathway are highly expressed in embryos and regulate neuronal positioning, whereas these molecules are expressed at low levels in adults and modulate synaptic plasticity. Reelin binds to Apolipoprotein E receptor 2 and Very-low-density lipoprotein receptors, triggers the phosphorylation of Disabled-1 (Dab1), and initiates downstream signaling. The expression of Dab1 marks neurons that potentially respond to Reelin, yet phosphorylated Dab1 is difficult to detect due to its rapid ubiquitination and degradation. Here we used adult mice with a lacZ gene inserted into the dab1 locus to first verify the coexpression of ß-galactosidase (ß-gal) in established Dab1-immunoreactive neurons and then identify novel Dab1-expressing neurons. Both cerebellar Purkinje cells and spinal sympathetic preganglionic neurons have coincident Dab1 protein and ß-gal expression in dab1(lacZ/+) mice. Adult pyramidal neurons in cortical layers II-III and V are labeled with Dab1 and/or ß-gal and are inverted in the dab1(lacZ/lacZ) neocortex, but not in the somatosensory barrel fields. Novel Dab1 expression was identified in GABAergic medial septum/diagonal band projection neurons, cerebellar Golgi interneurons, and small neurons in the deep cerebellar nuclei. Adult somatic motor neurons also express Dab1 and show ventromedial positioning errors in dab1-null mice. These findings suggest that: (i) Reelin regulates the somatosensory barrel cortex differently than other neocortical areas, (ii) most Dab1 medial septum/diagonal band neurons are probably GABAergic projection neurons, and (iii) positioning errors in adult mutant Dab1-labeled neurons vary from subtle to extensive.
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Encéfalo/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Neuronas/metabolismo , Médula Espinal/metabolismo , Animales , Encéfalo/crecimiento & desarrollo , Ratones , Proteínas del Tejido Nervioso/genética , Especificidad de Órganos , Proteína Reelina , Médula Espinal/crecimiento & desarrolloRESUMEN
INTRODUCTION: The reprogramming of a patient's somatic cells back into induced pluripotent stem cells (iPSCs) holds significant promise for future autologous cellular therapeutics. The continued presence of potentially oncogenic transgenic elements following reprogramming, however, represents a safety concern that should be addressed prior to clinical applications. The polycistronic stem cell cassette (STEMCCA), an excisable lentiviral reprogramming vector, provides, in our hands, the most consistent reprogramming approach that addresses this safety concern. Nevertheless, most viral integrations occur in genes, and exactly how the integration, epigenetic reprogramming, and excision of the STEMCCA reprogramming vector influences those genes and whether these cells still have clinical potential are not yet known. METHODS: In this study, we used both microarray and sensitive real-time PCR to investigate gene expression changes following both intron-based reprogramming and excision of the STEMCCA cassette during the generation of human iPSCs from adult human dermal fibroblasts. Integration site analysis was conducted using nonrestrictive linear amplification PCR. Transgene-free iPSCs were fully characterized via immunocytochemistry, karyotyping and teratoma formation, and current protocols were implemented for guided differentiation. We also utilized current good manufacturing practice guidelines and manufacturing facilities for conversion of our iPSCs into putative clinical grade conditions. RESULTS: We found that a STEMCCA-derived iPSC line that contains a single integration, found to be located in an intronic location in an actively transcribed gene, PRPF39, displays significantly increased expression when compared with post-excised stem cells. STEMCCA excision via Cre recombinase returned basal expression levels of PRPF39. These cells were also shown to have proper splicing patterns and PRPF39 gene sequences. We also fully characterized the post-excision iPSCs, differentiated them into multiple clinically relevant cell types (including oligodendrocytes, hepatocytes, and cardiomyocytes), and converted them to putative clinical-grade conditions using the same approach previously approved by the US Food and Drug Administration for the conversion of human embryonic stem cells from research-grade to clinical-grade status. CONCLUSION: For the first time, these studies provide a proof-of-principle for the generation of fully characterized transgene-free human iPSCs and, in light of the limited availability of current good manufacturing practice cellular manufacturing facilities, highlight an attractive potential mechanism for converting research-grade cell lines into putatively clinical-grade biologics for personalized cellular therapeutics.
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Células Madre Pluripotentes Inducidas/metabolismo , Animales , Diferenciación Celular , Reprogramación Celular , Expresión Génica , Genómica , Humanos , Ratones , TransgenesRESUMEN
Mutations in human LIS1 cause abnormal neuronal migration and a smooth brain phenotype known as lissencephaly. Lis1+/− (Pafah1b1) mice show defective lamination in the cerebral cortex and hippocampal formation, whereas homozygous mutations result in embryonic lethality. Given that Lis1 is highly expressed in embryonic neurons, we hypothesized that sympathetic and parasympathetic preganglionic neurons (SPNs and PPNs) would exhibit migratory defects in Lis1+/− mice. The initial radial migration of SPNs and PPNs that occurs together with somatic motor neurons appeared unaffected in Lis1+/− mice. The subsequent dorsally directed tangential migration, however, was aberrant in a subset of these neurons. At all embryonic ages analyzed, the distribution of SPNs and PPNs in Lis1+/− mice was elongated dorsoventrally compared with Lis1+/+ mice. Individual cell bodies of ectopic preganglionic neurons were found in the ventral spinal cord with their leading processes oriented along their dorsal migratory trajectory. By birth, Lis1+/− SPNs and PPNs were separated into distinct groups, those that were correctly, and those incorrectly positioned in the intermediate horn. As mispositioned SPNs and PPNs still were detected in P30 Lis1+/− mice, we conclude that these neurons ceased migration prematurely. Additionally, we found that a dorsally located group of somatic motor neurons in the lumbar spinal cord, the retrodorsolateral nucleus, showed delayed migration in Lis1+/− mice. These results suggest that Lis1 is required for the dorsally directed tangential migration of many sympathetic and parasympathetic preganglionic neurons and a subset of somatic motor neurons.
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1-Alquil-2-acetilglicerofosfocolina Esterasa/deficiencia , Movimiento Celular , Proteínas Asociadas a Microtúbulos/deficiencia , Malformaciones del Sistema Nervioso/metabolismo , Neuronas/patología , Médula Espinal/metabolismo , Médula Espinal/patología , 1-Alquil-2-acetilglicerofosfocolina Esterasa/genética , Animales , Animales Recién Nacidos , Sistema Nervioso Autónomo/citología , Sistema Nervioso Autónomo/metabolismo , Sistema Nervioso Autónomo/patología , Movimiento Celular/genética , Regulación hacia Abajo/genética , Femenino , Masculino , Ratones , Ratones Noqueados , Proteínas Asociadas a Microtúbulos/genética , Malformaciones del Sistema Nervioso/genética , Malformaciones del Sistema Nervioso/patología , Embarazo , Médula Espinal/citologíaRESUMEN
Reports based primarily on anatomical evidence suggest that olfactory ensheathing glia (OEG) transplantation promotes axon regeneration across a complete spinal cord transection in adult rats. Based on functional, electrophysiological, and anatomical assessments, we found that OEG promoted axon regeneration across a complete spinal cord transection and that this regeneration altered motor responses over time. At 7 months after transection, 70% of OEG-treated rats showed motor-evoked potentials in hindlimb muscles after transcranial electric stimulation. Furthermore, a complete spinal cord retransection performed 8 months after injury demonstrated that this axon regeneration suppressed locomotor performance and decreased the hypersensitive hindlimb withdrawal response to mechanical stimulation. OEG transplantation alone promoted reorganization of lumbosacral locomotor networks and, when combined with long-term training, enhanced some stepping measures. These novel findings demonstrate that OEG promote regeneration of mature axons across a complete transection and reorganization of spinal circuitry, both of which contribute to sensorimotor function.
Asunto(s)
Axones/fisiología , Miembro Posterior/fisiopatología , Neuroglía/trasplante , Bulbo Olfatorio/trasplante , Traumatismos de la Médula Espinal/terapia , Regeneración de la Medula Espinal/fisiología , Animales , Electrofisiología , Masculino , Actividad Motora/fisiología , Neuroglía/fisiología , Bulbo Olfatorio/fisiopatología , Estimulación Física , Análisis de Componente Principal , Ratas , Ratas Wistar , Recuperación de la Función/fisiología , Traumatismos de la Médula Espinal/fisiopatología , Estadísticas no ParamétricasRESUMEN
Spinal Wistar Hannover rats injected with olfactory ensheathing glia (OEG) have been shown to recover some bipedal stepping and climbing abilities. Given the intrinsic ability of the spinal cord to regain stepping with pharmacological agents or epidural stimulation after a complete mid-thoracic transection, we asked if functional recovery after OEG injections is due to changes in the caudal stump or facilitation of functional regeneration of axons across the transection site. OEG were injected rostral and caudal to the transection site immediately after transection. Robotically assisted step training in the presence of intrathecal injections of a 5-HT(2A) receptor agonist (quipazine) was used to facilitate recovery of stepping. Bipedal stepping as well as climbing abilities were tested over a 6-month period post-transection to determine any improvement in hindlimb functional due to OEG injections and/or step training. The ability for OEG to facilitate regeneration was analyzed electrophysiologically by transcranially stimulating the brainstem and recording motor evoked potentials (MEP) with chronically implanted intramuscular EMG electrodes in the soleus and tibalis anterior with and without intrathecal injections of noradrenergic, serotonergic, and glycinergic receptor antagonists. Analyses confirmed that along with improved stepping ability and increased use of the hindlimbs during climbing, only OEG rats showed recovery of MEP. In addition the MEP signals were eliminated after a re-transection of the spinal cord rostral to the original transection and were modified in the presence of receptor antagonists. These data indicate that improved hindlimb function after a complete transection was coupled with OEG-facilitated functional regeneration of axons. This article is part of a Special Issue entitled: Understanding olfactory ensheathing glia and their prospect for nervous system repair.
Asunto(s)
Axones/fisiología , Regeneración Nerviosa/fisiología , Neuroglía/trasplante , Bulbo Olfatorio/trasplante , Traumatismos de la Médula Espinal/cirugía , Vértebras Torácicas , Animales , Trasplante de Células/métodos , Trasplante de Células/fisiología , Terapia por Ejercicio/métodos , Neuroglía/citología , Neuroglía/fisiología , Bulbo Olfatorio/citología , Bulbo Olfatorio/fisiología , Ratas , Ratas Wistar , Recuperación de la Función/fisiología , Traumatismos de la Médula Espinal/patologíaAsunto(s)
Moléculas de Adhesión Celular Neuronal/metabolismo , Movimiento Celular/fisiología , Corteza Cerebral/citología , Proteínas de la Matriz Extracelular/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Neuronas/fisiología , Serina Endopeptidasas/metabolismo , Médula Espinal/citología , Animales , Corteza Cerebral/metabolismo , Ratones , Neuronas/citología , Proteína Reelina , Médula Espinal/metabolismoRESUMEN
Olfactory ensheathing glia (OEG) are distinct from other glia in their developmental origin, presence in both the peripheral and central nervous systems, and highly restricted location. OEG are present only in the olfactory lamina propria, olfactory nerve, and the outer two layers of the olfactory bulb, where they envelop bundles of olfactory sensory neuron axons in a manner distinct from myelination. Because of their unique properties and their association with the continually generated olfactory sensory neurons, OEG have attracted interest for their potential capacity to support axonal regeneration, for example, after spinal cord injury. However, study of the properties and function of OEG has been hampered by a paucity of neurochemical markers with which to identify and distinguish them definitively from other types of glia. Here we provide evidence through anatomical colocalization studies that OEG express the water channel aquaporin 1 (AQP1), both in vivo and in vitro. We propose that AQP1 expression represents an important distinguishing characteristic of OEG, which may impart unique function to these glia.
Asunto(s)
Acuaporina 1/metabolismo , Neuroglía/metabolismo , Bulbo Olfatorio/citología , Animales , Acuaporina 1/genética , Biomarcadores/metabolismo , Células Cultivadas , Femenino , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Masculino , Ratones , Ratones Noqueados , Neuroglía/citología , Bulbo Olfatorio/metabolismo , Mucosa Olfatoria/citología , Mucosa Olfatoria/metabolismo , RatasRESUMEN
Transplantation of olfactory bulb-derived olfactory ensheathing glia (OEG) combined with step training improves hindlimb locomotion in adult rats with a complete spinal cord transection. Spinal cord injury studies use the presence of noradrenergic (NA) axons caudal to the injury site as evidence of axonal regeneration and we previously found more NA axons just caudal to the transection in OEG- than media-injected spinal rats. We therefore hypothesized that OEG transplantation promotes descending coeruleospinal regeneration that contributes to the recovery of hindlimb locomotion. Now we report that NA axons are present throughout the caudal stump of both media- and OEG-injected spinal rats and they enter the spinal cord from the periphery via dorsal and ventral roots and along large penetrating blood vessels. These results indicate that the presence of NA fibers in the caudal spinal cord is not a reliable indicator of coeruleospinal regeneration. We then asked if NA axons appose cholinergic neurons associated with motor functions, i.e., central canal cluster and partition cells (active during fictive locomotion) and somatic motor neurons (SMNs). We found more NA varicosities adjacent to central canal cluster cells, partition cells, and SMNs in the lumbar enlargement of OEG- than media-injected rats. As non-synaptic release of NA is common in the spinal cord, more associations between NA varicosities and motor-associated cholinergic neurons in the lumbar spinal cord may contribute to the improved treadmill stepping observed in OEG-injected spinal rats. This effect could be mediated through direct association with SMNs and/or indirectly via cholinergic interneurons.