Retrovirally delivered Islet-1 increases recruitment of Ng2 expressing cells from the postnatal SVZ into the striatum.

Neural stem and progenitor cells hold the promise to be used in cell-based therapies to treat both acute and degenerative neurological diseases. To date, most research has been focused on the use of in vitro propagated stem cells used as a source of cells in cell replacement therapies. However, mobilization of endogenous neural stem cells to generate a specific differentiated cell type offers an attractive alternative. In this study, we investigate the possibility to direct the formation of specific cells from the endogenous stem and progenitor cells residing in the subventricular region of the postnatal brain. With the aim to induce postnatal generation of striatal neurons, we ectopically expressed Islet-1, a LIM homeodomain transcription factor expressed by striatal progenitors during development, in cells of the subventricular zone (SVZ) of neonatal and adult rats. Ectopic expression of Islet-1 in the neonatal, but not adult, SVZ resulted in the appearance of a population of cells in the striatum. These cells were primarily located in the ventrolateral area of the striatum where they differentiate into Ng2 expressing cells. However, no neurogenesis was observed in the striatum, nor was ectopic striatal differentiation observed in any other area of the brain after retroviral expression of Islet-1 in the SVZ. Thus, although ectopic expression of Islet-1 is sufficient to direct the migration of cells into the striatum in neonatal animals, it does not specify a striatal projection neuron phenotype in cells generated from the SVZ after birth.

Neurogenin2 identifies a transplantable dopamine neuron precursor in the developing ventral mesencephalon.

In neural transplantation studies, there is an interest in identifying and isolating mesencephalic dopamine (mesDA) neuron precursors that have the capacity to differentiate into fully mature mesDA neurons after transplantation. We report here that in the developing ventral mesencephalon (VM) the proneural gene Neurogenin2 (Ngn2) is expressed exclusively in the part of the ventricular zone that gives rise to the migrating mesDA neuroblasts, but not in the differentiated mesDA neurons. From other studies, we know that Ngn2 is involved in the generation of mesDA neurons and that the development of mesDA neurons is severely compromised in Ngn2-null mutant mice. We show here that cells isolated by FACS from the developing VM of Ngn2-GFP knock-in mice are capable of generating mesDA neurons, both in vitro and after transplantation to the striatum of neonatal rats. All mesDA neuron precursors, but not the serotonergic or GABAergic neuron precursors, are contained in the Ngn2-GFP-expressing population. Moreover, all glial cells were generated from cells contained in the GFP-negative cell fraction. The results show that surviving mesDA neurons in VM grafts are derived from early postmitotic, probably Nurr1-expressing precursors before they have acquired their fully differentiated neuronal phenotype. The Ngn2-GFP reporter construct used here thus provides a tool for the identification of mesDA neuron precursors in the VM and selective isolation of transplantable mesDA neuron precursors for transplantation.

Strengths and limitations of the neurosphere culture system.

After the initial reports of free-floating cultures of neural stem cells termed neurospheres (1,2), a wide array of studies using this promising culture system emerged. In theory, this was a near-perfect system for large-scale production of neural cells for use in cell replacement therapies and to assay for and characterize neural stem cells. More than a decade later, after rigorous scrutiny and ample experimental testing of the neurosphere culture system, it has become apparent that the culture system suffers from several disadvantages, and its usefulness is limited for several applications. Nevertheless, the bulk of high-quality research produced over the last decade has also shown that under the right circumstances and for the appropriate purposes, neurospheres hold up to their initial promise. This article discusses the pros and cons of the neurosphere culture system regarding its three major applications: as an assay for neural stem cells, as a model system for neurogenesis and neural development, and for expansion of neural stem cells for transplantation purposes.

Striatal neuron differentiation from neurosphere-expanded progenitors depends on Gsh2 expression.

Neural stem and progenitor cells from the embryonic forebrain can be expanded under growth factor stimulation in vitro, either as free-floating aggregates called neurospheres or as attached monolayer cultures. We have previously shown that despite the maintenance of important regulatory genes such as Gsh2, in vitro expansion of cells from the lateral ganglion eminence (LGE) restricts their differentiation potential. Specifically, their ability to differentiate into striatal projection neurons is compromised. It is not clear whether this restriction is caused by loss of progenitors with the ability to generate striatal projection neurons or whether the restricted differentiation potential is caused by factors lacking during in vitro differentiation. To address this, we have set up an in vitro system, in which expanded LGE-derived cells are differentiated in coculture with primary cells isolated from different regions of the embryonic brain. We provide evidence that the primary cells supply the expanded cells with contact-mediated region-specific developmental cues. Neurosphere-expanded LGE progenitors can, when presented with these cues, differentiate into neurons with characteristics of striatal projection neurons. Furthermore, we show that the ability of the expanded LGE cells to respond to the developmental cues presented by the primary cells depends on the maintained expression of Gsh2 in the expanded cells.