RESUMEN
A continuous normal function of olfactory ensheathing glia (OEG) is to promote axonal regeneration from the olfactory neuroepithelium to the brain, and their neuroregenerative potential in other CNS sites such as the injured spinal cord has been studied for over a decade. However, human OEG are difficult to obtain in large amounts directly from tissues, and the derived primary cultures have a limited duplication capacity. Thus, although auto-transplantation may be an obvious option for initial proof-of-concept trials, alternatives must be explored to obtain large quantities of homogeneous, pre-characterized OEG for wide-scale therapeutic use. We have cultured primary human OEG derived from olfactory bulbs (OB) obtained by necropsy and successfully extended the replicative lifespan of these cells using lentivectors encoding Bmi-1 and TERT transgenes flanked by loxP sites. In contrast to the primary cells which could only be expanded for a limited number of passages (approximately 12), adult human OEG immortalized Bmi-1/TERT divided indefinitely in culture. Clonal lines were isolated and the floxed transgenes could be excised by lentivector-mediated Cre recombinase delivery. Primary, immortalized, and deimmortalized human OEG all expressed typical markers of this cell type and importantly, were all able to promote axonal regeneration of adult rat retinal ganglion neurons (RGN) in co-culture assays.
Asunto(s)
Regeneración Nerviosa/fisiología , Neuroglía/fisiología , Bulbo Olfatorio/citología , Adolescente , Adulto , Animales , Animales Recién Nacidos , Células Cultivadas , Células Clonales , Técnicas de Cocultivo/métodos , Femenino , Proteínas Fluorescentes Verdes/genética , Humanos , Masculino , Proteínas Asociadas a Microtúbulos/metabolismo , Persona de Mediana Edad , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Neuroglía/trasplante , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Complejo Represivo Polycomb 1 , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Ratas , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Células Ganglionares de la Retina/metabolismo , Traumatismos de la Médula Espinal/cirugía , Telomerasa/genética , Telomerasa/metabolismo , Transducción Genética/métodosRESUMEN
Immortalized cell lines of olfactory ensheathing glia (OEG) that maintain the proregenerative properties of primary cultures provide an unlimited source of OEG for both basic and applied studies. Indeed, one specific immortalized rat OEG clonal line (TEG3) proved to be as good as primary OEG in promoting neuritogenesis and axon regeneration in culture models. Thus, we examined the capacity of TEG3 to promote axonal repair in an animal model of spinal cord injury, dorsal column crush. TEG3 cells can acquire astrocyte-like or Schwann cell-like morphology depending on the conditions under which they are cultured. In the injured spinal cord, prelabeled TEG3 survived for at least 10 weeks after grafting and they integrated into the spinal cord, adopting Schwann cell-like, astrocyte-like, or intermediate morphologies. In TEG3-transplanted animals, sensory projection axons grow into the lesion site and there was robust sprouting/axonal growth of the corticospinal tract, both into and beyond the lesion site, after crushing of the spinal cord-dorsal columns. TEG3-transplanted animals also recovered sensory and motor function in tape removal and beam walking behavioral tests. These data indicate that certain immortalized cell lines derived from a single cell can maintain the regenerative properties of primary OEG.
Asunto(s)
Células Clonales/trasplante , Neuroglía/trasplante , Bulbo Olfatorio/citología , Traumatismos de la Médula Espinal/fisiopatología , Traumatismos de la Médula Espinal/terapia , Animales , Axones/metabolismo , Línea Celular Transformada , Ratas , Médula Espinal/patología , Médula Espinal/fisiologíaRESUMEN
Olfactory bulb ensheathing glia (OEG) have attracted special attention during the last few years because of their unique properties in promoting regeneration of adult mammalian central nervous system (CNS) components. However the molecular and cellular characteristics responsible for this capacity remain to be revealed. Such studies are presently hindered by the lack of a plentiful source of homogenous OEG. Thus the availability of immortalized OEG lines maintaining the regenerative characteristics of the primary cultures would represent an unlimited source of OEG for use not only in biochemical analyses of neuroregenerative mechanisms but also to characterize their regenerative properties in models in culture and in vivo. We have immortalized primary rat OEG using the SV40 large T antigen expressed from a constitutive cellular promotor, and report here the isolation and characterization of clonal lines. These OEG clonal lines were comparable to primary OEG and Schwann cells in the promotion of axonal regeneration of mature rat retinal ganglion neurons (RGN) but, significantly, this culture assay system more closely reflects the in vivo reparative properties of OEG on transected nerves than other assays of neuritogenesis in that it revealed OEG cells to promote the growth of a larger number of long axons than Schwann cells. Using this assay we were able to grade our OEG lines for their neuroregenerative capacity, opening the possibility of identifying molecules with correlative expression levels in these cells. Our preliminary characterization revealed that the expression level of a classical OEG marker, the p75-NGF receptor, does not correlate with neuroregenerative capacity.
Asunto(s)
Regeneración Nerviosa/fisiología , Neuroglía/citología , Neuroglía/fisiología , Bulbo Olfatorio/citología , Células Ganglionares de la Retina/citología , Animales , Antígenos de Diferenciación/biosíntesis , Axones/fisiología , Western Blotting , Células Cultivadas , Células Clonales/citología , Técnicas de Cocultivo , Inmunohistoquímica , Ratas , Células Ganglionares de la Retina/metabolismo , Células de Schwann/citologíaRESUMEN
During all the life of a mammal, olfactory ensheathing glia (OEG) permit the entry and navigation of olfactory neuron axons from peripheral to central nervous system (CNS) territory. This physiological characteristic of OEG has been successfully used for promotion of axonal regeneration after CNS injury in animal models. However, cellular and molecular properties responsible for OEG regenerative ability remain to be unveiled. Two approaches may be followed: to carry out genomic or proteomic analysis to detect secreted and/or membrane bound molecules or to examine the expression of molecules previously described as neuritogenic. This is the case of amyloid precursor protein (APP), a neurite-promoting molecule. We have studied the expression of APP by OEG and OEG-derived clonal lines, immortalised with the large T antigen of SV40 (TEG lines). OEG express high levels of APP in vivo and in culture. TEG lines maintained high expression of APP. Western blot analysis showed the presence of high molecular weight forms of APP in OEG, corresponding probably to glycosylated forms and/or to higher expression of the full length APPs. The main APP isoforms present in OEG cultures were APP770 and 751. L-APP isoforms without the exon 15, which are those corresponding with proteoglycan forms, are predominant in glial cells. Our data showed that OEG had three times as much L-APP as astrocytes, which may correlate with OEG neuritogenic capacity. In conclusion APP, a neurite-promoting molecule, is produced by OEG. Its nexin activity, dependent on the Kunitz family of serine protease inhibitors (KPI) domain and/or in combination with its glycosylation level might contribute with other factors to the ability of these cells to foster axonal elongation.