Directed differentiation of adult neural stem cells for cell therapy in the nervous system

Sammanfattning: The emergence of neural stem cell research has raised hopes for cell therapy of neurological disorders. Insight into the normal mechanisms of neural stem cell fate specification and differentiation is a prerequisite for the development of safe and efficient clinical applications. As the pathology of each neurological disorder is determined by damage to a specific neural cell population, it will be necessary to develop protocols for the generation of specific neural cell types. These neurons and/or glial cells may therefore need to be tailored through directed differentiation, rather than grafting naïve neural stem cells. Paper I: We have developed protocols to introduce genes into adult neural stem cells using viral and non-viral vectors in vitro and in vivo. Adenoviral and VSV G-pseudotyped retroviral vectors were more efficient than VSV G-pseudotyped lentiviral vectors and plasmid transfection in vitro. Plasmids could be delivered in vivo when complexed with polyethyleneimine, and expression could be targeted to neural stem/progenitor cells by the use of specific promoters in the expression constructs. We could further show that these methods can be used for directed differentiation of adult neural stem cells in vitro. Ectopic expression of the neuronal determination factor, neurogenin2, inhibited the generic, mainly astroglial fate and directed transduced cells to a neuronal fate, as indicated by expression of the neuronal marker betaIII-tubulin and neuronal cell morphology. Paper II: We have analyzed the possibility of using developmentally relevant genes in directed differentiation to generate specific neuronal subtypes from adult neural stem cells. We produced expression constructs encoding transcription factors (MNR2, Nkx6.1) previously shown to promote the induction of ectopic motor neuron markers (Islet1, Islet2, Lim3 and Hb9) when ectopically expressed in the developing neural tube. We confirmed such induction following electroporation into the chick neural tube. We analyzed the effect of these transcription factors on neurosphere cultures derived from adult mouse and rat lateral ventricle and spinal cord and mouse embryonic day 9.5 neural tube.Neither MNR2, nor Nkx6.1 could promote induction of motor neuron markers in these cell cultures. However, coexpression of MNR2 and neurogenin2 reproducibly induce expression of Islet1 in neurosphere cultures of all included sources. Islet1 is an early motor neuron marker but is also expressed in other cell types. The Islet1 positive cells were characterized to be negative for other motor neuron markers (Lim 1, Lim3, VAChT, and Peripherin). These data indicate that neural stem cells in culture differ in competence to respond to embryonic cues, compared to progenitor cells in the developing neural tube. Paper III: We investigated the feasibility of using directed differentiation of adult neural stem cells ex vivo, followed by transplantation to the adult mammalian nervous system. The cochlear sensory epithelium and spiral ganglion neurons in the adult mammalian inner car do not regenerate following severe injury. To replace the degenerated cells, neural stem cells could be a source for replacement cell therapy. In order to direct the differentiation of grafted cells towards a neuronal fate, neural stem cells were retrovirally transduced with a construct encoding neurogenin2 prior to transplantation. Surviving cells in animals receiving neurogenin2transduced neural stem cells expressed the neuronal marker betaIII-tubulin. Transplanted cells were found close to the sensory epithelium as well as adjacent to the spiral ganglion neurons and their peripheral processes. Similar neuronal induction was achieved from grafting naïve neural stem cells to the chemically deafened inner ear, but not to the intact inner ear. The study lends support to the notion that it may be possible to design a cell therapy strategy based on neural stem cells for die replacement of degenerated or absent cochlear neurons. Paper IV: We used directed differentiation of adult neural stem cells ex vivo prior to transplantation to a rat model of spinal cord impact injury. Several studies have reported functional improvement after transplantation of neural stem cells to the injured spinal cord. We provided evidence that grafting of naïve adult neural stem cells into a rat thoracic spinal cord weight drop injury improves motor recovery but also causes aberrant axonal sprouting associated with allodynia-like hypersensitivity of forepaws. Directed differentiation of neural stem cells with neurogenin2 suppresses astroglial differentiation of engrafted cells, prevents graft-induced sprouting, alleviating allodynia and promotes further functional recovery. The low grade of neuronal differentiation observed was surprising as neurogenins are potent stimulators of neuronal differentiation in vitro, in addition to their inhibition of an astrocytic fate. However, environmental cues also influence the fate of neurogenin expressing cells. The present data suggest that a non-neurogenic environment in the spinal cord largely overrides the neurogenic effect of neurogenin2 and instead the engrafted cells are more prone to acquire an oligodendroglial fate. Graft-derived oligodendrocytes were associated with increased amounts of myelin at the lesion site and with improved skillful hindlimb motor function. These findings show that neural stem cell transplantation to the injured spinal cord have positive effects on recovery, but can cause severe side effects. Our findings indicate that controlled differentiation of neural stem cells may be required for safe clinical application. The findings also stress that an analysis of allodynic symptoms should be included in primate studies prior to the clinical application of neural stem cells. Conclusion: Our findings show that it is possible to direct the differentiation of adult neural stein cells in cell culture ex vivo and that such modifications also affect the differentiation of the cells following transplantation to the nervous system and can influence functional recovery.