WO2008124863A1 - Method of generating population of cells enriched for undifferentiated cells - Google Patents

Abstract

The invention relates to generation of population enriched for undifferentiated cells based on the level of expression of doublecortin. There is also provided in population of DCX+ve cells enriched for undifferentiated cells comprising cells which are DCXlow and methods for the use of such a population of cells.

Description

METHOD OF GENERATING POPULATION OF CELLS ENRICHED FOR

UNDIFFERENTIATED CELLS

Technical Field

The present invention relates to a population of cells enriched for undifferentiated cells and methods for generating such a population. More particularly, the present invention relates to a population of cells enriched for undifferentiated cells and methods for generating such a population wherein cells which express doublecortin at a low level are selected.

Background Art

Doublecortin (DCX) is a 40 kDa microtubule- associated protein that is expressed in a precise temporal manner in migrating neuroblasts during early embryonic development. It is also retained in neurogenic areas in the adult (the subventricular zone/ rostral-migratory stream/ olfactory bulb axis and the dentate gyrus of the hippocampus) , a pattern that suggests it is an ideal marker of both embryonic corticogenesis and adult neurogenesis (Francis et al., 1999; Rao and Shetty, 2004; Couillard-Despres et al . , 2005). High DCX expression occurs transiently during the early stages of corticogenesis, with the highest levels of expression being observed at embryonic day 14, before decreasing to low levels by birth (des Portes et al . , 1998; Francis et al., 1999; Hannan et al., 1999; Meyer et al . , 2002; Brown et al., 2003; Lee et al . , 2003). Moreover, the protein is not re-expressed during gliogenesis or regenerative axonal growth (Couillard-Despres et al . , 2005), confirming its selectivity for newly born neurons and not later neuronal regenerative events. The restriction of DCX expression to the neuronal precursor phase of the neuronal lineage has also been further suggested (Brown et al . , 2003) by its lack of co-localization with nestin, a marker of multipotential precursors.

The lack of useful stage-specific markers has made the characterization of lineage specific cell types difficult. However, the generation of transgenic mice expressing reporter genes such as green fluorescent protein (GFP) under the control of cell type specific promoters now provides a source of cells suitable for lineage interrogation both in vivo and in vitro. Examples include the neural precursor-specific markers nestin

(Frisen et al . , 1995) and glial fibrillary acidic protein

(GFAP) (Nolte et al., 2001), the pan-neuronal marker tubulin _1 (Gloster et al . , 1994), the neuronal precursor specific pro-opiomelanocortin (Overstreet et al . , 2004) and the oligodendroglial marker CNP (Gravel et al . , 1998).

Summary of the Invention

In this study, we have used two strains of DCX- GFP transgenic mice to isolate and characterize DCX^+ve cells. This has revealed that virtually all cells expressing high levels of DCX are restricted to the neuronal lineage, whereas the population that expresses low levels of DCX contains a significant number of multipotential precursors.

Accordingly, in a first aspect the present invention provides a population of DCX^+ve cells enriched for undifferentiated cells which are DCX^low.

In an embodiment the cells are β III-tubulin^"ve and/or MAP 2^"ve.

In an embodiment the cells are PSA and/or NCAM^+ve. In an embodiment the cells are nestin^+ve. In an embodiment the cells are neurosphere- forming cells. The population of cells may be derived from the embryonic or adult brain regardless of developmental stage or brain region. The enriched population includes at least some multipotent cells. Typically the population of undifferentiated cells includes stem cells, and more preferably pluripotent neural stem cells. Proliferation and/differentiation may occur, and therefore the population may include undifferentiated progeny or differentiated cells derived from the undifferentiated cells .

According to a further aspect of the present invention there is provided a method of generating a population of DCX^+ve cells enriched for undifferentiated cells from a biological sample, comprising the steps of: (i) disrupting the biological sample to provide a mixed population comprising the undifferentiated cells and other cells;

(ii) sorting the mixed population into populations based on the level of expression of doublecortin; and (iii) selecting DCX^low cells to generate said population of DCX^+ve cells enriched for undifferentiated cells. According to a further aspect of the present invention there is provided a method for cell replacement therapy in an animal, said method comprising providing a population of cells as described above, and introducing said population to an animal in need of such treatment. According to a further aspect the present invention provides a composition for cell replacement therapy wherein the composition comprises a population of cells as described above and an inert carrier.

In an embodiment the method involves the replacement of neural or non-neural tissue in an animal by introducing said population to said animal .

Additionally, the present invention provides a method for the treatment of neurodegenerative disease in an animal, said method comprising providing a population of cells as described above and introducing said population to said animal .

The method of the invention may further comprise inducing differentiation and proliferation.

Accordingly, in a further aspect the invention provides a composition comprising a population of DCX^+ve cells enriched for undifferentiated cells comprising cells which are DCX^low , or a cell population generated from said undifferentiated cells by induction of differentiation and proliferation, and a medium capable of supporting the growth of the cells.

The population of undifferentiated cells of the present invention and progeny thereof are also useful as a model for the screening of potential neurologically reactive therapeutic compounds.

Accordingly, in a further aspect of the present invention there is provided a method of screening potentially neurologically active therapeutic compounds comprising the steps of:

1) providing a composition comprising a population of cells as dscribed above and a medium capable of supporting the growth of the cells; 2) contacting said cell population or progeny thereof with at least one candidate pharmaceutical agent; and

3) determining if said candidate agent has an effect on proliferation, differentiation, survival, phenotype or function of said cell population.

Brief Description of the Drawings

Figure 1. DCX and GFP expression co-localize in DCX-GFP transgenic mouse lines. In transgenic DCX-GFP/2kb brain sections GFP^+ve cells are found in the E14 cortex and thalamus (A, B and C) and in the cortex (D, E and F) in a pattern that colocalized with DCX staining. In the adult DCX-GFP/BAC line, GFP expression was observed in the rostral migratory stream and olfactory bulb (H and K) in a pattern that coincides with native DCX staining (G, I and

J) . Scale bars represent lmm in A, B, C, J and K and lOOμm in D -I.

Figure 2. GFP relative fluorescence intensity correlates with DCX expression levels in individual cells. Cells from adult DCX-GFP/BAC SVZ were isolated and stained with a DCX-specific antibody. Flow cytometry analysis revealed a strong correlation between the GFP relative fluorescence intensity and the level of DCX immunostaining in individual cells. In all cases, cells that expressed GFP were also DCX^+ve, with no false positives being observed. Figure 3. FACS isolation and neurosphere activity of E14 DCX-GFP/2kb populations: (A) Single viable cells were isolated from E14 GFP^+ve brains using FACS and were sorted into four populations (high, mid, low and negative) based on DCX expression. Unsorted cells from a wild-type littermate were collected as a control (B) . The DCX^low population was enriched for neurosphere formation, while the number of neurospheres grown from the DCX^mid population

(C) was significantly reduced. Virtually no neurospheres were detected in the DCX^high population (***p<.001; n=4, when compared to unsorted control) .

Figure 4. Neurosphere activity of P2 DCX^+ve cells. Higher neurosphere-forming activity was observed in the DCX^+ve population from whole P2 DCX-GFP/2kb brains compared to DCX^"ve and unsorted cells (A; n=3) . Within this DCX^+ve population, however, the higher expressing cells (DCX^mid) had a lower neurosphere-forming frequency compared to the cells with lower (DCX^low) expression (B; n=2) . Overall, the P2 cerebellum had relatively high levels of DCX expression and was the only region in which a significantly higher number of neurospheres was observed in the DCX^low cells compared to the DCX^"ve population (C; *p<0.05, n=3) .

Figure 5. Neurosphere activity of P2 cerebellum cells. The DCX^high cells in the P2 DCX-GFP/2kb cerebellum had a significantly lower neurosphere-forming frequency then the DCX^"ve cells in this region (***p<0.001, n=4) , while the DCX^low population was enriched for neurosphere- forming cells.

Figure 6. Precursor properties of DCX^+ve cells from the adult SVZ (A) and hippocampus (B) of the DCX- GFP/BAC strain. (A) Neurosphere assays revealed that neurosphere- forming frequency of the DCX^low population in the adult SVZ was similar to that of the DCX^"ve population (n=4) .

(B) The DCX^low population in the adult hippocampus had a significantly higher neurosphere-forming frequency than the DCX^"ve and unsorted populations (*p<0.05, n=4) .

Figure 7. Precursor properties of DCX^+ve cells in the adult DCX-GFP/BAC olfactory bulb. The olfactory bulb had the highest levels of DCX expression in the adult brain. This DCX^+ve population had a lower neurosphere- forming frequency than the DCX^"ve population (A) . Of this DCX^+ve population, the DCX^hi9h cells had no neurosphere activity while the DCX^low cells were enriched for neurosphere formation (B) . Figure 8. Electrical properties of cortical DCX^+ve cells in acute forebrain slices of E14 and P2 DCX-GFP/2kb mice. (A) Scatter plots of resting membrane potential

(RMP) and input resistance (Rin) of cortical E14 (n=18) and P2 (n=24) DCX^+ve cells. Asterisks denote a significant difference between E14 and P2 groups (**p<0.01). (B) Representative traces of voltage responses to current injection in two different DCX^+ve cells. Examples of cells in which an action potential (AP) was either absent (E14) or evoked (P2) in response to depolarizing current pulses. (C) . Bar graphs of normalized populations of E14 (n=18) and P2 (n=24) cells in which an AP (exhibiting an overshoot positive to 0 mV) either could or could not be evoked.

Figure 9. Depolarization-activated whole-cell currents recorded from cortical DCX^+ve cells of E14 and P2 DCX-GFP/2kb mice. (A) Representative family of membrane currents recorded from a P2 DCX^mid cell in response to depolarizing voltage steps. Holding potential, -60 mV. Insert: Transient inward currents displayed at higher gain. (B) Membrane currents recorded from a P2 DCX^mid cell in response to a voltage step from -60 mV to 0 mV in the absence (i) and presence of 300 nM TTX (ii) and 300 Nm TTX + 10 mM TEA (iii) . A TTX-sensitive Na+ channel current trace (iv) was obtained by subtracting traces (i) and (ii) . Similarly, a TEA-sensitive K+ channel current trace (v) was obtained by subtracting traces (ii) and (iii) . (C and D) Current-voltage (I-V) relationships obtained for transient inward Na+ current and persistent outward K+ current in cortical E14 and P2 DCX^+ve cells. Cells were divided into two groups based on their maximum inward current densities: those exhibiting currents <10 pA/pF (•) and those with transient inward currents >10 pA/pF (•) .

Figure 10. Passive and active electrical properties of SVZ and dentate gyrus DCX^low cells in adult DCX-GFP/BAC brain slices. (A) Scatter plots of resting membrane potential (RMP) and input resistance (Rin) of DCX^low cells in the SVZ (n=10) and the dentate gyrus (DG; n=12) and DCX^'ve cells in the dentate gyrus (n=6) . Asterisks denote a significant difference between SVZ and DG DCX^low cells (**p<0.01). (B) Representative traces of action potentials (APs) induced by current injection in DCX^low and DCX^"ve cells obtained from the dentate gyrus. Examples of cells which exhibited either a single (DCX^low) or repetitive (DCX^"ve) AP. (C) . Bar graphs of normalized populations of SVZ (n=10) and dentate gyrus (n=12) DCX^low cells and dentate gyrus (n=6) DCX^"ve cells in which an AP (exhibiting an overshoot positive to 0 mV) either was or was not evoked. (D and E) Current-voltage (I-V) relationships obtained for transient inward Na+ current and persistent outward K+ current at a holding potential of -80 mV in DCX^low cells in the SVZ (n=5) and the dentate gyrus (n=ll) . Cells were divided into two groups: those exhibiting maximum inward current densities <10 pA/pF (•) and those exhibiting current densities >10 pA/pF (•) .

Figure 11. Neurosphere activity of E14 and P2 sorted cells from the DCX-GFP/BAC transgenic line. (A) Single viable cells were isolated from E14 GFP^+ve brains using FACS and were sorted into four populations (high, mid, low and negative) based on GFP expression. Unsorted cells from a wild-type littermate were collected as a control. The DCX^high and OCX^mid populations had significantly reduced numbers of neurospheres compared to the DCX^low and unsorted control (***p<0.001, n=4) . (B) In P2 brains lower neurosphere-forming frequencies was observed in the DCX^mid compared to the cells with lower DCX expression (DCX^low) (n=3) .

Detailed Description of the Preferred Embodiments The present invention relates to the enrichment of a population of DCX^+ve cells for undifferentiated cells such as stem cells which are capable of differentiation into multiple mature cell lineages. In particular, the present invention provides undifferentiated cells that may be used in transplantation strategies for the replacement or repair of degenerate tissue. The ability to now generate enriched populations of undifferentiated cells further enables the identification of factors which may be used to stimulate proliferation, differentiation and self- maintenance of undifferentiated cells in vivo. The present invention is particularly directed to neural stem cells (NSCs) and neural progenitor and precursor cells with the capacity to differentiate into cells and cell lineages required for the development, maintenance, or repair of tissue associated with a central nervous system in animals, mammals and humans. The present invention further provides methods for the enrichment of undifferentiated cells which are capable of proliferation and differentiation into distinct functional progeny which constitutes the functional cells of the CNS. Cell types that can be derived from the undifferentiated cells of the present invention include but are not limited to neurons, oligodendrocytes, glia and astrocytes. The subject invention further contemplates the use of undifferentiated cells for the repair or regeneration of tissue associated with the CNS, as well as tissue augmentation, gene therapy and therapeutic drug targeting in an animal or a human. The undifferentiated cells of the present invention are further useful in a method for repair and/or regeneration of tissue in an animal or a mammal such as a human. The instant invention is further useful in transplantation and differentiation of undifferentiated cells in an animal or a mammal such as a human. The present invention provides a method for the prophylaxis and treatment of neurodegenerative disorders and to correct neurological dysfunction and/or trauma. Furthermore, the undifferentiated cell cultures prepared in accordance with the present invention are useful in screening for molecules which can influence the growth and differentiation of undifferentiated cells. Such molecules are referred to as endogenous activators . The use of endogenous activators avoids the need for transplantation of undifferentiated cells including stem cells into a subject.

Reference herein to a "population" of cells means two or more cells. A population may comprise one or more cell types. A "cell type" may be cells of the same lineage or sub-type having substantially the same physiological status .

As used herein the term "enriched" in the context of a cell population or a reference to an "enriched population" , "enriched cell population" or any equivalent term means that the cell population includes a greater number of cells with a particular characteristic compared to the equivalent population which has not been the subject of a selection process. The term "disruption" or "disrupting" includes disassociation of individual cells from the connecting extracellular matrix (ECM) of a tissue constituting or forming part of a biological sample. Preferably, a single cell suspension is produced. Preferably, the individual cells are of a minimal size such as from about 5 to about 50 microns or from about 7 to about 30 microns or from about 7 to about 20 microns. Most preferably, the cells are greater than about 12 microns .

"Undifferentiated" means a primordial state of a cell or cells capable of differentiation and proliferation to produce progeny cells that can be physiologically/ biochemically, morphologically, anatomically, immunologically, physiologically, or genetically distinct from the primordial state. As stated above, the preferred undifferentiated cells are stem cells and are more preferably NSCs which can give rise to multiple cell lineages, are capable of differentiation and proliferation and are capable of self-renewal . The most preferred NSCs give rise to neuronal cells. The NSCs may be pluripotent NSCs or non-pluripotent NSCs or a mixture of both. A "biological sample" is a tissue or organ or part thereof, such as from the different regions of the brain or nervous tissue. A "sub-sample" may be a portion of a tissue, such as a biopsy or a part or portion of an organ or a tissue. An in vitro culture of cells is also regarded as a biological sample.

The present invention provides, therefore, a method of producing a population of DCX^low undifferentiated cells which are capable of giving rise to multiple cell lineages. This provides a means for gene therapy, augmentation therapy and therapy to repair, replace and to delay senescence in neural and non-neural tissue. It also enables identification of growth factors and other agents which can promote proliferation and/or differentiation of endogenous cells. Reference herein to "cell replacement therapy" includes, in one form, a process in which undifferentiated cells are strategically placed in vivo or in vitro such as to differentiate and proliferate into a particular cell lineage or into multiple cell lineages. Thus, cell replacement therapy requires that an undifferentiated cell appropriately differentiates for the purposes of providing repair, regeneration or replacement of a cell function including the replacement of an organ or a tissue. ^ΛλCell replacement therapy" also includes augmentation therapy. The latter includes the removal of existing cells or tissue, expanding in culture and then replacing. The subject into which the undifferentiated cells or their progeny are implanted for the purpose of "cell replacement therapy" or repair of tissue, or from which undifferentiated cells can be derived, is preferably an animal including but not limited to animals such as cows, pigs, horses, chickens, cats, dogs and is preferably a mammal such as a primate and most preferably a human.

By "tissue" is meant a part of an organism consisting of a number of cells having a similar structure or a similar function. For example, neural tissue may consist of a number of cells comprising one or more cell type including but not limited to neurones, glia, oligodendrocytes, astrocytes and ependymal cells. Organs are considered herein to comprise tissue and a brain is encompassed by the term organ, and a sub-sample includes a biopsy of a tissue such as but not limited to nervous tissue or an organ such as but not limited to a brain.

The present invention is directed to methods of the enriching for non-terminally differentiated cells or undifferentiated cells, such that the differentiation of the cell is not inhibited without destroying the ability of the cell to proliferate. As used herein, "pluripotent cells" shall mean any non-terminalIy differentiated cells. The undifferentiated cell is preferably an NSC.

The undifferentiated cells in the population of the present invention are in a viable state and, hence, have potential in various therapeutic protocols especially in the treatment of CNS disorders.

CNS disorders encompass numerous afflictions such as neurodegenerative diseases (e.g. Alzheimer's disease and Parkinson's disease), acute brain injury (e.g. stroke, head injury, cerebral palsy) and a large number of CNS dysfunctions (e.g. depression, epilepsy, and schizophrenia) . In recent years, neurodegenerative disease has become an important concern due to the expanding elderly population which is at greatest risk for these disorders. These diseases which include Alzheimer's disease, multiple schlerosis (MS), Hungtington' s disease, amyotrophic lateral schlerosis and Parkinson's disease, have been linked to the degeneration of neural cells in particular locations of the CNS, leading to the inability of these cells or the brain region to carry out their intended function. Degeneration in a brain region known as basal ganglia can lead to diseases with various cognitive and motor symptoms, depending on the exact location. The basal ganglia consists of many separate regions, including the striatum (which consists of the caudate and putamen) , the globus pallidus, the substantia nigra, substantia innominate, ventral pallidum, nucleus basalis of Meynert, ventral tegmental area and the subthalamic nucleus. Preferably, differentiation of undifferentiated cells can replace damaged neural and/or non-neural tissue. Preferably, the undifferentiated cells are used in cell replacement therapy, tissue augmentation therapy, gene therapy, amongst other methodologies . Alternatively, growth factors, identified using cultures enriched for undifferentiated cells, are used to stimulate proliferation and/or differentiation of cells in vivo.

A number of cell isolation, cell separation and cell purging strategies are known for purifying or removing cells from a suspension comprising a diverse population of cells. Cell separation methods are used to isolate cells or purge cell suspensions or cell populations typically fall into one of three broad categories. Physical separation methods typically exploit differences in a physical property between cell types, such as cell size or density (e.g. centrifugation or elutriation) ; chemical-based methods typically employ an agent which selectively kills or purges one or more undesirable cell types; and affinity-based methods typically exploit antibodies or molecules with a selective binding capacity which bind selectively to marker ligands on a cell membrane surface of desired or undesired cell types, which antibodies may subsequently enable the cells to be isolated or removed from the suspension.

As described herein, the undifferentiated cell populations generated by the subject methods have a range of utilities. Other utilities contemplated by the present invention include putting a marker on or within an undifferentiated cell and then using this to trace the undifferentiated cell and/or its progeny throughout the body and to monitor its physiological changes. This provides an ability to direct therapies to multiple targets. The enriched populations of undifferentiated cells are also useful for gene and protein discovery and in particular the mechanisms involved in differentiation and proliferation. This enables the therapeutic production of new neurons in various disease states.

Furthermore, the undifferentiated cells of the present invention are useful in cytokine delivery via, for example, endogenous stem cell activation and for selective migration of undifferentiated cells and their progress to sites of injury.

The undifferentiated cells may also be used to screen for molecules which can influence the growth, proliferation and/or differentiation of stem cells. This information allows therapeutics to be developed which can be used to activate the endogenous undifferentiated cell to differentiate into new neurons or glia and the like. Thus, this aspect of the present invention does not require transplantation to occur. Such molecules would, however, be an important adjunct to successful transplantation therapy.

Generation of the population of the present invention allows for methods for screening drug candidates for effectiveness in increasing neurogenesis. Screening assays may be performed directly using a culture. Candidate agents may be initially screened for the ability to modulate neurogenesis through its effect on an in vitro culture. For example, in a method which involves contacting the candidate drug and the culture of the present invention, the effect on differentiation and proliferation of the undifferentiated cell population may be observed, but equally the effect on survival, phenotype or function of these cells or their progeny could be observed. An in vivo drug screening or drug discovery process involving engrafting a non-human mammal with an enriched population of neural stem cells is described in United States Patent No. 7,105,150, the contents of which were incorporated herein by reference. The engrafted non- human mammal is useful for drug screening and drug discovery using well known methodology. Methods for screening a candidate agent against a cell culture are described, for example, in United States Patent No. 7,041,438 using methods well known in the art. Assessment of the activity of candidate agents generally involves combining a cell culture with a candidate compound, determining any resultant change, and then correlating the effect of the compound with the observed change.

In addition, differentiated cells arising from the culture of the present invention can be used for tissue reconstitution or regeneration in a human patient in need thereof. The cells are administered in a manner that permits them to graft to the intended tissue site and reconstitute or regenerate the functionally deficient areas. Thus, the in vivo transplantation process comprises implanting members of the undifferenticated cell population of the present invention into a mammal once this population has been treated with one or more growth factors to induce differentiation, for example, once they have been induced to differentiate into neurons and/or glia. In vitro proliferation and differentiation of neural stem cells is described, for example, in United States Patent No. 7,115,418, the contents of which are incorporated herein by reference. The growth factors necessary to induce proliferation and/or differentiation are well known to the person skilled in the art and include, but are not limited to, NGF, BDNF, the neurotrophins, CNTF, amphiregulin, FGF-I, FGF-2, EGF, TGFα, TGFβ, PDGF, IGFs and the interleukins .

EXAMPLE 1

A construct containing 2kb of the mouse DCX promoter, from -1802 bp to +199 bp relative to the translation start site in the plasmid pBluescript, was obtained from Prof. Peter Jeffrey (Children's Medical Research Institute, Sydney, Australia) . A fragment containing the enhanced GFP gene (eGFP) and the SV40 poly A was removed from plasmid pEGFPl (Clontech/ BD Biosciences, Heidelberg, Germany) by digestion with Sail and AfIII and blunt-ending the AfIII site with Klenow. Unless otherwise noted, all enzymes were obtained from New England Biolabs (Frankfurt, Germany) . The fragment containing the eGFP gene was cloned into Sail and

BfrBIdigested pBS-DCX to generate the clone pDCXeGFP.

Plasmid DNA was purified using the plasmid maxiprep kit

(Qiagen Australia, Melbourne, Australia) , after which the

DCXeGFP cassette was removed by digestion of 40 μg of DNA with Spel, BsrBX and XmnX . Digested DNA was run on a 0.7% TBE gel and the insert purifed using the Qiagen gel purification kit. Transgenic mice (DCX-GFP/2kb) were generated by pronuclear injection of the DNA cassette into fertilized BCBFl mouse eggs, which were then implanted into pseudopregnant animals. DNA was isolated from the resulting offspring, and founder mice were identified via polymerase chain reaction (PCR) using primers DCXP2009F

(5 ' -CAGAACCAGAACCTTGCAGGC-3 ' ) and eGFPR2 (5' -

GCGCGGGTCTTGTAGTTGCCG- 3'). The PCR conditions were 1 cycle of 94°C for 3 mins, followed by 40 cycles of 94°C for 30 sec, 60⁰C for 30 sec and 72°C for 30 sec, with a final cycle of 72⁰C for 7 mins. A second DCX-GFP transgenic mouse (DCX-GFP/BAC) was obtained from the Mutant Mouse Regional Resource Center (MMRRC) , Gensat BAC transgenic project (Gong et al . , 2002). Lineages of founder mice were established by breeding transgenic animals with wildtype C57B16 mice. Animals were treated in accordance with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes, and all experiments were approved by the University of Queensland Animal Ethics Committee.

Immunohistochemical analysis of GFP and DCX expression in transgenic tissue sections

Experiments were performed on day 14 embryos (E14) and postnatal day 2 (P2) pups using either the DCX- GFP/2kb or DCX-GFP/BAC transgenic strain, whereas those experiments performed on adult tissue made use of the DCX- GFP/BAC animal. Adult C57B16 mice were time-mated to heterozygous DCX-GFP transgenic animals. The brains from E14 embryos or P2 pups were immediately dissected and fixed in 4% parafomaldehyde (PFA) in 0. IM phosphate buffered saline (PBS) for 3 hours at 4⁰C. Adult animals were perfused with 0. IM PBS followed by 4% PFA. The brains were then removed and incubated overnight in 4% PFA, followed by a further overnight incubation in 15% sucrose in 0.1M PBS. They were subsequently incubated in 1:1 (v:v) 15% sucrose: optimal cutting temperature compound (OCT) and then in 100% OCT before being snap frozen in OCT and stored at -80⁰C. Coronal and sagittal sections (15μm) were cut on a cryostat (Leica Microsystems GmbH, Wetzlar, Germany) and collected onto glass slides. Sections were postfixed in 4% PFA for 5 mins and washed briefly in PBS before being incubated for 60 minutes at room temperature with blocking solution: 5% fetal calf serum (FCS; JHR Biosciences, Brooklyn, Australia) plus 5% normal goat serum (NGS; Sigma-Aldrich, Sydney, Australia) in 0. IM PBS containing 0.1% X-100 Triton (Sigma-Aldrich). The blocking solution was then replaced with fresh blocking solution containing guineapig anti-DCX antibody (1:200; Chemicon,

Temecula, CA) and the sections incubated either for 60 minutes at room temperature or at 4⁰C overnight, after which they were washed with PBS and incubated for 40 minutes at room temperature in blocking solution containing Alexa Fluor 546 goat anti-guinea-pig antibody

(1:1000; Molecular Probes/Invitrogen, Eugene, OR) and

4¹ ,6-diamidino-2-phenylindole (DAPI; 1:1000; Molecular

Probes/Invitrogen) . Following washing with PBS, the slides were coverslipped with fluorescence mounting medium (DakoCytomation, Carpinteria, CA) before viewing on an Olympus upright fluorescence microscope. Images were captured by a digital camera linked to a computer running analysis software (Olympus Australia, Brisbane, Australia) .

Fluorescence-activated cell sorting and generation of primary neurosphere and neuronal cultures

E14, P2 and adult brains were collected as described above and processed either whole or dissected into specific regions (olfactory bulb, cortex, cerebellum and subventricular zone (SVZ) ) . GFP^+ve cells were separated by fluorescence-activated cell sorting (FACS) using a FACS Vantage cell sorter (BD Biosciences) . A wild-type littermate control was used to determine background fluorescence levels. For primary neurosphere cultures the cells were plated at a density of 500 cells/well in 96- well plates (Falcon/BD Biosciences, San Jose, CA) with 0.2 ml complete medium per well. Complete medium consisted of mouse NeuroCult™ NSC Basal Medium plus mouse NeuroCult™ NSC Proliferation Supplements (StemCell Technologies, Vancouver, Canada) with 2% bovine serum albumin (Roche, Basel, Switzerland) and 2 μg/ml heparin (Sigma-Aldrich) . The following growth factors were also included: 20 ng/ml purified mouse receptor-grade epidermal growth factor

(EGF; BD Biosciences Australia) and 10 ng/ml recombinant bovine basic fibroblast growth factor (FGF-2; Roche) . Primary cells were incubated for 7 days in humidified 5% CO2 to permit neurosphere formation. The primary neurospheres were then counted and collected for passaging or differentiation. Results of the neurosphere counts were expressed as mean + standard error and statistical analysis was performed using a standard t-Test (two sample assuming equal variance) .

Neurosphere passaging and differentiation Neurospheres were collected after 7 days and passaged as previously described (Bull and Bartlett, 2005) . The passaged cells were then re-plated with 2ml complete medium at a density of 20000 cells/well in 24- well tissue culture plates (Falcon) . Neurospheres were passaged every 7 days for up to 12 passages, after which they were differentiated for 5 days then stained for the neuronal marker βlll-tubulin and the astrocytic marker GFAP with a DAPI counterstain (see below) .

Immunocytochemistry of DCX-GFP cells and FACS analysis

Brains were dissected from E14, P2 or adult DCX- GFP mice, then dissociated and resuspended in 875μl cold PBS . Cells were fixed by incubation with 62.5μl cold 4% PFA for 30 mins at 4⁰C. They were then pelleted by centrifugation at 104 rcf for 7 mins and permeabilized using 1 ml PBS containing 0.2% Tween (Sigma-Aldrich) with an incubation period of 15 mins at 37⁰C. After blocking for 30 mins at 4⁰C in PBS containing 2% FCS, the cells were incubated in primary antibodies: βlll-tubulin (1:1000, mouse monoclonal; Promega, Madison, WI), MAP2ab

(1:250, mouse monoclonal; Neomarkers, Fremont, CA), nestin

(1:250, mouse monoclonal; Chemicon, Rosemont, IL), Ki67

(1:250, mouse monoclonal; BD Pharmingen, San Diego, CA),

Dlx2 (1:250, rabbit polyclonal; Chemicon), GFAP (1:500, rabbit polyclonal/ DakoCytomation) or DCX (1:250, guinea pig polyclonal; Chemicon) for 30 mins at 4⁰C. Primary antibody was removed by washing the cells once in 1 ml blocking solution (2% FCS, 2%NGS, 0.1% Triton X-100) before incubating at 4⁰C for 30 mins in 200μl of secondary antibody (1:1000 dilution of anti-mouse PE, BD Biosciences or anti-rabbit-PE, Molecular Probes) . The cells were then washed in PBS before being resuspended in 1 ml PBS for analysis by FACS. Cells stained with secondary antibody alone were used as a background control for determining the negative gates. For PSA-NCAM staining cells were incubated for 30 mins in anti-PSA-NCAM (1:500, mouse monoclonal/ Chemicon) , then washed and incubated for 30 mins in AlexaFluor 633 anti-mouse IgM (1:700; Invitrogen/

Molecular Probes) . Cells were analyzed using a FACS

Vantage cell sorter (BD Biosciences) or an ImageStream 100

(Amnis, Seattle, WA) .

Electrophysiological methods Acute brain slice preparation

Whole brains were dissected and placed in ice- cold, 95%02/5%C02-saturated artificial cerebrospinal fluid (aCSF) composed of (in mM) : 126 NaCl, 2.5 KCl, 2.4 CaCl2, 1.2 MgCl2, 1.2 Na2HPO4, 26 NaHCOa and 10 glucose at pH 7.4. Coronal forebrain (E14, P2 and adult >3 weeks) or hippocampal (adult > 3 weeks) slices of 300-400 μm thickness were cut in cold aCSF using a vibratome (Vibroslice; World Precision Instruments, Sarasota, FL) . Slices were then stored in oxygenated aCSF at room temperature for >1 hr to allow recovery.

Electrophysiological recordings DCX-GFP^+ve cells were identified in acute brain slices. Whole-cell current and voltage clamp recordings were obtained from these cells using an Axopatch 200B patch clamp amplifier (Molecular Devices, Union City, CA) . Data were acquired at 5-10 kHz and filtered at 2-5 kHz with a Digidata 1320 interface (Molecular Devices) , linked to a personal computer equipped with pCLAMP 9.0 software (Molecular Devices) . Whole-cell capacitance was compensated and leak currents were subtracted during voltage clamp recordings. The liquid junction potential was not corrected. Patch electrodes, which were pulled from borosilicate glass capillaries, had resistances of 1.5-4 MΩ when filled with pipette solution. For slice patch recordings, a coronal forebrain slice was placed in a submersion recording chamber, where it was held in position by nylon threads attached to a U-shaped platinum wire and continuously perfused with oxygenated aCSF at a rate of ~5 ml/min. Dialyzed wholecell patch clamp recordings were performed using a pipette solution containing (in inM) : 130 KCl, 5 NaCl, 0.4 CaCl2, 1 MgCl2, 1.1 EGTA and 10 HEPES-NaOH at pH 7.3. For dialyzed or perforated patch clamp recordings from cultured DCX^+ve cells, cells attached to a glass coverslip were placed in a recoding chamber and continuously perfused at a rate of ~1 ml/min with the following extracellular recording solution (in mM) : 140 NaCl, 3 KCl, 2 CaCl2, 1 MgCl2, 10 HEPES-NaOH, and 10 glucose at pH 7.4. The pipette solution for cultured DCX^+ve cells contained (in mM) : 145 KCl, 5 NaCl, 10 HEPES-NaOH, pH adjusted to 7.2. For perforated patch clamp recordings, the patch electrode was filled with an intracellular solution containing 240 g/ml amphotericin B. However, there was no apparent difference in the electrical properties of cells recorded under either dialyzed or perforated patch configurations. Experiments were performed at room temperature (E14 and C (adult) . Cell capacitance (CDP2) or 30-32m) was measured by integrating the capacitative transient evoked during a 10 mV depolarizing step from a holding potential of -70 mV. Resting membrane potential (RMP) and input resistance (Rin) were determined from current clamp recordings in the absence and during a hyperpolarizing current pulse of 10- 200 pA, respectively. Data were analyzed using Clampfit 9.2 and Prism 4.0 (GraphPad, San Diego, CA). Results

GFP expression co -localizes with native DCX expression in brain of embryonic and postnatal DCX-GFP transgenic mice

Of the eleven founder lines examined for the DCX- GFP/2kb transgenic strain, only one displayed high levels of GFP expression. To ensure this GFP expression colocalized with DCX protein expression, sections were stained with an anti-DCX antibody. At E14, we found that Qpp^+ve QQiI_s predominantly coincided with DCX staining in the neopallatial cortex (the future cerebral cortex) , the diencephalon (thalamus) and the intermediate zone of the lateral and medial ganglionic eminences (striatum) (Fig. IA-F) . Strong co-expression was also observed in the embryonic spinal cord. No expression or staining was observed in either the ventricular zone of the telencephalon or the ventricular zone of the ganglionic eminence. In postnatal sections, the level of GFP expression was lower than that observed in the embryo but was again restricted to regions stained with DCX antibody. There was extensive labeling in the cortical plate and the intermediate zone regions, as well as in the hippocampus and olfactory bulb, with strong expression in the cerebellum. However, in the DCX-GFP/2kb transgenic strain, no GFP expression or staining with an anti-GFP antibody could be detected in the adult (data not shown) . This led us to examine a second strain DCX-GFP/BAC in which GFP was inserted into a BAC clone (RP23-462G16) just upstream of the DCX coding region. This animal had strong GFP expression in the dentate gyrus of the hippocampus, the SVZ, the rostral migratory stream and the olfactory bulb

(Fig. IH and K) , and this expression was found to co- localize with immunostaining for DCX (Fig. IG, I and J) .

GFP intensity reflects the level of DCX expression in individual cells

To confirm that the GFP intensity accurately reflected the level of DCX expression in individual cells, cells were isolated from both transgenic strains, stained with a DCX specific antibody and analyzed by FACS. For the DCX-GFP/2kb transgenic mouse, cells from E14 whole brain were analyzed while for the BAC strain cells from the adult SVZ were used. Analysis of this co-labeling using the ImageStream 100 clearly showed that the GFP intensity accurately reflected the level of endogenous DCX expression in single cells (Fig 2) . Cells with high DCX expression also expressed high levels of GFP protein, but, more importantly, all cells lacking GFP expression showed an absence of DCX staining and no false GFP positives were detected.

Defining the characteristics of DCX-expressing cells at different developmental stages and in different brain regions

We examined the DCX-expressing populations at various stages of development in order to determine whether the characteristics of cells expressing similar DCX levels varied with age or had some common features.

At E14 , DCX -expression levels define a neuronally restricted population

To determine the lineage restriction and proliferative potential of DCX^+ve cells during early neurogenesis, GFP^+ve cells were isolated from E14 brains from both the DCXGFP/2kb and DCX-GFP/BAC lines by FACS. At this developmental age these cells comprised approximately 40% of the total population. For further analysis, the E14 DCX^+ve cell population was gated into three equal populations; these were designated high, mid and low based on DCX expression (Fig. 3A) . For each experiment, unsorted cells from a wild-type littermate were run through the flow cytometer as a control (Fig. 3B) . To assess the percentages of freshly isolated E14 DCX^+ve cells that were already expressing cell type-specific markers or were proliferating, these cells were stained with antibodies directed against βlll-tubulin, MAP2, NeuN, nestin and Ki- 67 and analyzed by FACS. Of the DCX^high population, 99.9% of cells co-expressed the neuronal marker βlII-tubulin, while 82% expressed MAP2. Consistent with this differentiated phenotype, a very low percentage of E14 DCX^+ve cells were mitotically active, with only 1% of DCX^+ve cells co- expressing the proliferation marker Ki- 67. There was some co-labeling of DCX with nestin (approximately 20% of E14 DCX^+ve cells) ; however this only occurred in the E14 DCX^low population, with no such colabeling seen in the E14 DCX^hl9h cells. No obvious staining of any E14 population was observed with NeuN. To study the precursor potential of the different populations, cells were collected and plated at 500 cells/well in neurosphere-supporting medium, and the number of neurospheres generated in each condition was counted after 7 days. The E14 DCX^high population from the

DCX-GFP/2kb line contained virtually no cells capable of forming neurospheres (0.03+0.02 spheres/500 cells). The

DCX^m:Ld population contained some neurosphere-forming cells

(0.39+0.18 spheres/500 cells), but the number was considerably lower than for unsorted cells (2.61+0.7 spheres/500 cells) or the DCX^low population (2.1±0.28 spheres/500 cells) , which both had a similar frequency of neurosphere-forming cells (Fig. 3C) . Similar results were obtained using the DCX-GFP/BAC transgenic line (DCX^high: 0.025±0.005 spheres/500 cells, DCX^mid: 0.06±0.001 spheres/500 cells, DCX^low: 0.18±0.001 spheres/500 cells, DCXve:0.08±0.002 spheres/500 cells and unsorted: 0.11+0.007 spheres/500 cells; see Figure 11) .

To determine whether the precursors that gave rise to neurospheres had stem cell-like (self-renewal) properties, neurospheres generated from the sorted E14 DCX^mid, DCX^low and DCX^"ve cell populations, as well as from unsorted cells, were passaged as bulk cultures for at least 12 passages. This resulted in a 10- to 40-fold expansion between passages, consistent with some of the precursors having stem cell-like properties. In addition, upon differentiation, neurospheres derived from all populations contained both neurons and astrocytes .

DCX*^ve cells from P2 brains have lower levels of expression and higher multipotential precursor activity than those from E14 brains.

At P2, DCX^+ve cells comprise approximately 30% of the total brain cell population. For further experiments, the P2 population from whole brain was divided into DCX^+ve and DCX^"ve cells. The levels of GFP expression at P2 were comparable to those obtained in the E14 DCX^mid and DCX^low populations. In contrast to our findings in the embryo, where there is a significant level of co-localization of DCX and neuronal markers, only 6% of the DCX^+ve cells in the postnatal brain expressed βlll-tubulin, whereas 13% expressed MAP2. Similar to what was found at E14, at P2 a very low percentage of DCX^+ve cells were mitotically active, with only 1% of DCX^+ve cells co-expressing the proliferation marker Kϊ 61. At P2 approximately 6% of DCX^+ve cells were positive for NeuN, a marker of more mature neurons. P2 DCX^+ve cells from whole brains had only mid to low levels of DCX expression but a higher neurosphere-forming 4.9 spheres/500 cells vs . _φ frequency than unsorted cells (175%, M=28.6 ± M=16.5±3.1 spheres/500 cells). The neurosphere- forming frequency was higher in both these conditions than in the DCX^"ve population (M=16.2+1.4 spheres/500 cells; Fig. 4A) . The higher expressing cells from the P2 whole brain (DCX^mid) , however, had a lower neurosphere-forming frequency (M=6.2+4.2 spheres/500 cells) than the DCX^low population (M=19.4+10.2 spheres/500 cells) (Fig. 4B). Once again similar results were seen using the DCX-GFP/BAC line

(DCX^mid: 2.34±0.38 spheres/500 cells, DCX^low: 4.44±1.7 spheres/500 cells, DCX^"ve: 0.22+0.06 spheres/500 cells, and unsorted: 2.6±0.42 spheres/500 cells. When the DCX^+ve cells were stained with PSA-NCAM, cells co-expressing both markers had a significantly higher neurosphere-forming frequency (M=15.21+3.8 spheres/500 cells) than the cells negative for these proteins (M=4.31+0.7 spheres/500 cells) . Unsorted cells that stained PSA-NCAM_^+ve also showed significantly higher neurosphere-forming frequency (M= 9.9±9.6 spheres/500 cells) than the PSA-NCAM^'ve population (M=3.7±1.0 spheres/500 cells).

A small population of DCl?^igh cells are found in P2 cerebellum

To further characterize which brain region contained the most DCX^+ve cells at P2, GFP^+ve brains were removed and various regions (olfactory bulb, cerebellum, cortex and SVZ) were dissected. DCX^+ve cells accounted for approximately 1% of the total number of cells in the P2 olfactory bulb, 8% of cells in the cortex, 16% of cells in the cerebellum and 2% of cells in the SVZ. The cerebellum contained a DCX^high population, with cells over an order of magnitude brighter than those in the other regions. The DCX^+ve cells in the SVZ and olfactory bulb only expressed relatively low levels of GFP. Neurosphere assays revealed that DCX^+ve cells from the cerebellum had a higher neurosphere-forming frequency (1.7±0.6 spheres/500 cells), than that of the unsorted cerebellum cells (0.9+0.6 spheres/500 cells; Fig. 4C) . However, the precursor activity of the DCX^high cells from the cerebellum (approximately 3% of the total population) was only 3.4 ±1.6% of that of the DCX^low population (Fig. 5). The DCX^{mid' low} cells in the olfactory bulb (0.8+0.3 spheres/500 cells) and cortex (17.6+2.9 spheres/500 cells) also had similar neurosphere activity to the corresponding DCX^'ve and unsorted cells (Fig. 4C) . An increase in neurosphere activity was also observed in the SVZ, which displayed a 1.5-2 fold increase in neurospheres in the DCX^low population (28.0+2.9 spheres/500 cells) when compared to the DCX^"ve cells (16.2+1.2 spheres/500 cells) (Fig. 4C) . DCX^low cells in the adult SVZ and hippocampus show high precursor activity while DCX^^13h cells from the olfactory bulb have low precursor activity

In the adult SVZ and hippocampus, cells express low levels of DCX, with these DCX-expressing cells comprising approximately 25% of the total cell population. For neurosphere assays, DCX^low and DCX^"ve cells were collected from the DCXGFP/BAC transgenic line. DCX^low cells from the SVZ region had a similar neurosphere-forming activity to that of the DCX^'ve and unsorted populations

(Fig. 6A) , while the DCX^low cells from the hippocampus had a significantly higher neurosphere-forming activity than the

DCX^"ve and unsorted populations (Fig. 6B) . The adult olfactory bulb had both the highest level of DCX expression and the most DCX^+ve cells (~ 70% of total olfactory bulb cells were DCX^+ve) . The DCX^high cells in the olfactory bulb had a lower neurosphere-forming frequency than the DCX^low and DCX^'ve populations (Fig. 7A & B) .

Membrane electrical properties of DCX*^ve cells in E14 and P2 mouse brain

To determine whether the DCX^hl3h cells which uniformly expressed βlll-tubulin also had the electrical properties of neurons, we performed whole-cell patch clamp recordings in slices from E14 and P2 forebrains . DCX^hi9h cells were investigated from the E14 cortices and DCX^mid cells from P2 cortices. Interestingly, almost all the DCX^hxgh cells in the E14 cortex were round and lacked processes, whereas most of the DCX^mid cells in P2 cortex had bipolar radial processes. As shown in Fig. 8A,DCX DCX^hxgh high cells from E14 cortical slices had the following resting membrane properties: RMP =-46.4 + 3.2 mV and Rin = 1533 ± 304 MΩ (n=18; mean ± SEM) . In comparison to DCX^high cells in E14 mice, DCX^mxd cells in P2 mice had an equivalent mean RMP of -46.8 ± 2.2 mV and lower mean Rin of 634 ± 117 MΩ (n=24) . Changes in membrane potential of DCX^+ve cells in response to current injection were examined under current clamp conditions. Almost none of the E14 DCX^high cells studied (only 1 out of 18 cells) exhibited an action potential (overshoot positive to 0 mV) in response to depolarizing current injection (Fig. 8B) . Although a similar small population of the P2 DCX^mid cells (2 out of 24 cells) exhibited a clear action potential (Fig. 8C) , an additional 3 cells exhibited an immature action potential

(a distinguishable spike but no overshoot) . Nevertheless, the majority of the population was neuronally immature/ non-excitable cells. To examine depolarizationactivated membrane currents, cells were held at -60 mV and ionic currents were elicited by depolarizing step pulses under voltage clamp conditions. All of the E14 DCX^hl3h and P2 DCX^mxd cells examined exhibited a persistent outward current with or without a transient inward current (Fig. 9A) . As shown in Fig. 9B, the transient inward current was completely inhibited by 300 nM tetroddtoxin (TTX) , indicating a Na+ channel current. Interestingly, TTX also enhanced the persistent outward current, suggesting that the TTX-sensitive current of DCX^+ve cells was a combination of a well-defined transient component and a persistent inward component (Fig. 9B(ii) and (iv) ) . Tetraethylammonium largely suppressed the persistent outward current, suggesting that it was mediated by a K₊ channel current. Analysis of current-voltage relationships indicated that cells were divided into two groups based on the magnitude of transient inward Na+ current densities . There were two distinguishable populations, particularly in P2 DCX^mid cells: those exhibiting inward Na+ current densities <10 pA/pF and those with Na₊ current densities

>10 pA/pF. The size of the two populations for both E14 and P2 DCX^+ve cells was similar (Fig. 9C and D) . Taken together, both E14 DCX^high and P2 DCX^mid cells represented mixed neuronal progenitor populations in terms of their membrane electrical properties. P2 DCX^mid cells exhibited greater diversity with regard to action potential characteristics and transient Na+ current; however, in both cases approximately half of the cells exhibited immature neuronal differentiation with small Na+ current density.

.Membrane electrical properties of DCX^+ve cells in adult mouse brain

_DCχ ^iow _ce]_j_s without any processes in the adult SVZ exhibited a mean RMP of -25.4 + 1.6 mV and a high mean Rin of 2993 ± 506 M_ (n=10; Fig.10A) . These cells also failed to produce an action potential in response to current injection (Fig.10C). In contrast, process-bearing DCX^low cells located in the subgranular zone or inner granule cell layer of the adult dentate gyrus exhibited a significantly more hyperpolarized mean RMP of -75.1 ± 10.0 mV and lower Rin of 825 ± 139 MΩ (n=12 ; Fig.10A) compared to those observed in the SVZ. A single action potential was elicited in two of twelve DCX^low cells in the dentate gyrus (Fig.1OB and C) in response to current injection. All DCX^"ve granule cells (non-labeled cells) in the dentate gyrus recorded as a control from the same slice preparations exhibited single or repetitive action potentials (Fig.1OB and C). Voltage-gated Na+ and K₊ currents were also examined in DCX^low cells in the SVZ and dentate gyrus. As shown in Fig.10D, all examined DCX^low cells in the SVZ exhibited only marginal inward Na+ current densities (<10 pA/pF) in accordance with cells not exhibiting an action potential. Outward K₊ current densities obtained from these cells were also less pronounced. In contrast, the majority (7 of 11 cells) of DCX^low cells in the dentate gyrus exhibited large Na₊ current densities (>10 pA/pF) , whereas the remainder had small Na+ current densities, indicating substantial diversity of the cell population in terms of Na+ current densities (Fig.10E). Outward K+ current densities of DCX^low cells in the dentate gyrus were independent of their Na+ current densities and were larger than those observed in DCX^low cells in the SVZ. Discussion

The value of DCX as a marker of migrating neuronal precursor cells both during development and in adult neurogenic regions has been well documented (Cooper- Kuhn and Kuhn, 2002; Brown et al . , 2003; Rao and Shetty, 2004) . However, until now the expression of DCX in relation to progenitor and stem cell activity has not been thoroughly investigated. Prior to the present study, the precise potential of DCX^+ve cells has been difficult to investigate directly due to the intracellular nature of the DCX protein. This has restricted both the isolation of DCX^+ve cells by flow cytometry for in vitro studies and the ability to record electrophysiologically from these cells both in slice preparations and in situ. To overcome this, we used two transgenic mouse lines expressing GFP under the control of the DCX promoter. The GFP expression pattern in both transgenic lines coincided with native DCX protein expression when examined immunohistochemically, a fact we confirmed using flow cytometric analysis of individual cells. Crucially, the latter demonstrated that the expression of GFP accurately reflects that of DCX, even at low levels. This was of particular importance in defining the DCX^low population that we found to contain multipotential precursor activity. Both lines gave similar findings in relation to the association of DCX levels to neuronal and multipotential precursor activity, the only difference being that the transgenic line in which GFP was driven off the 2kb DCX promoter region lacked GFP expression in the adult brain. The finding that adult DCX expression is driven differentially to that of the embryo and early postnatal animal is of particular interest. A previous study identified several putative transcription factor binding sites in the 3.5 kb DCX regulatory region studied (Karl et al . , 2005). Many of these binding sites, including Brn-2, NeuroDl, E2F-1 and E2F-2, are known to be involved in neuronal development. While the promoter region we used in the DCX-GFP/2kb line contained most of these transcription factor binding sites, one potential binding site previously identified further upstream was not included. The absence of the E2F-1 binding site in our DCX-GFP/2kb transgenic mice could potentially lead to a decrease in the expression of GFP in the adult, as mice bearing a targeted deletion of E2F-1 have been shown to have reduced neurogenesis in the adult CNS, while the numbers of neurons in the neocortex, which develops exclusively during embryogenesis, is unchanged (Cooper- Kuhn and Kuhn, 2002) . In contrast, the DCX-GFP transgenic line generated using the BAC construct, uses a large genomic region upstream of the DCX gene to drive GFP expression and therefore this promoter is likely to contain all the elements required for faithful expression of the GFP protein. It has been widely accepted that cells expressing high levels of DCX are neuronally committed (Brown et al., 2003). The most surprising finding from the present flow cytometry studies was, therefore, that the DCX^+ve cell population contained not only cells restricted to the neuronal lineage but also a sizeable population of neurosphere-forming precursors which had the hallmarks of stem cells: self-renewal and multipotentiality. Furthermore, these two types of cells could be differentiated from each other on the basis of DCX expression levels, regardless of developmental stage or brain region. While not expected, these findings are not incompatible with previous reports, which have based their conclusion on results obtained in immunohistochemically processed tissue in which only cells expressing relatively high levels of DCX can be visualized. In the present study, such cells, for example the DCX^high population in the E14 forebrain, were also found to be exclusively neuronal in lineage, with 99.9% expressing the neuronal marker βlll-tubulin. They also appeared to be largely postmitotic as revealed by the low frequency of Ki-67-positive cells (<1%) . It has previously been shown that the highest levels of DCX expression are usually found in migrating neurons, suggesting an immature phenotype, which is consistent with the function of DCX as a regulator of microtubular formation (Francis et al., 1999). Electrophysiological studies using slice preparations from the E14 cortex showed that the DCX^high cells were indeed predominantly immature neuronal cells, as they had either a complete absence of Na⁺ current or a Na⁺ current density insufficient to produce an action potential. However, neurons were found at various stages of neuronal maturation, from those without Na⁺ currents through to those in which an action potential could be clearly identified. At P2, when developmental neuronal migration is complete and DCX expression levels are lower, the cells had increased Na⁺ current density, as would be expected of a more mature population. Nevertheless, the P2 population did contain a number of more immature cells with low to absent Na+ current. These presumably reflect the neurosphere-forming precursor cells. In adult preparations, the membrane electrical properties of DCX^low cells depended on the brain region in which they were located. The depolarized RMP and high Rin observed for _Dcχ ^iow j-eiis i_n the SVZ are consistent with those reported for neuronal progenitors (βIII-tubulin^+ve) in the SVZ and posterior rostral migratory stream (Wang et al . , 2003; Liu et al., 2005) but different from those described for GFAP+ve progenitors in the SVZ, which exhibit a hyperpolarized RMP of ~ -80 mV and low Rin of ~50 MΩ (Liu et al . , 2005) . The depolarized RMP may be attributed in part to a voltage error caused by the high Rin as reported previously (Wang et al . , 2003). None of the DCX^low cells in the SVZ exhibited an action potential or distinct Na+ current densities of >10 pA/pF. The membrane electrical properties of DCX^low cells in the dentate gyrus were significantly different from those of the DCX^low cells in the SVZ. In contrast to functionally differentiated granule cells, which exhibit repetitive action potentials in response to depolarizing current injection, only 17% of the DCX^low cells elicited an action potential. Together with the variation in Na+ current densities in these cells, DCX^low cells in the dentate gyrus appear to represent early to intermediate stage neuronal progenitors, similar to DCX^mid cells in the P2 cortex. In contrast, DCX^low cells in the SVZ comprise a relatively uniform population of early stage neuronal progenitors. As outlined above, the finding that the DCX^low population contained a significant number of neural stem-like cells that gave rise to neurospheres, which could be subsequently passaged extensively and shown to differentiate into multiple neural types, was unexpected. Even more surprising was the finding that in the P2 ventricular zone and cerebellum, as well as in the adult SVZ, hippocampus and olfactory bulb, the DCX^low population contained a significant number of neurospheres compared to the DCX^'ve population. Although it has previously been reported that there is a population of DCX^+ve cells in the dentate gyrus and the SVZ that expresses the proliferation marker Ki-67 (Brown et al . , 2003), these cells were not thought to be multipotential precursors as there was no co-localization of DCX with nestin or GFAP

(Couillard-Despres et al . , 2005). Our results, however, demonstrate the expression of nestin in 20% of DCX^low cells at E14, further reflecting the stem-like nature of some of these cells. These results suggest that many of the DCX^low cells are not, as previously thought, committed to the neuronal lineage but are in fact multipotential neural precursors. The fact that these cells could be repeatedly passaged over 10 generations further suggests that they are not restricted in their selfrenewal capacity. Whether most of the stem cells in the brain go through a stage of DCX expression prior to final commitment to the neuronal lineage, or whether this represents an entirely separate precursor population, remains to be resolved. However, the fact that these cells are found in many brain regions at many stages of development suggests that this is an important sub-population that requires further characterization. The presence of neurosphere-forming cells in the cerebellum within the DCX^low population contradicts the findings of an earlier study, which reported that the multipotential cerebellar precursor is not within the neuroblast lineage (Lee et al . , 2005). In fact, our data suggest that nearly all the stem-like cells in the cerebellum reside in the DCX^low population. This discrepancy does not appear to be due to different markers being used to define the neuronal lineage cells, since we show here that PSA-NCAM, one of the markers used in the aforementioned study to exclude neuronal lineage cells, was found to be co-expressed with DCX on a large proportion of the neurosphere-forming cells from the postnatal brain. One possible explanation of this discrepancy is that the previous study excluded all cells expressing Math-1, so it would be interesting to determine if this population contained the DCX^low, PSA-NCAM^+Ve stem cells. The adult olfactory bulb contained DCX^high cells, which have previously been reported to be the granular and periglomerular neurons (Nacher et al . , 2001). However, while this could suggest recent migration into the bulb, such an assumption is not consistent with the observation of lower levels of DCX in the rostral migratory stream. One possibility is that DCX, because of its role in regulating cytoskeletal changes, is involved in the neural plasticity occurring in the olfactory bulb, including neurite outgrowth and synaptogenesis (Nacher et al . , 2001) . However, this seems unlikely since DCX is absent from regions such as the hypothalamus, where continuous synaptic reorganization occurs and other markers of synaptic plasticity, such as PSA-NCAM, are expressed

(Alonso et al., 1997). In summary, this study has allowed us to divide DCX^+ve cells in the brain into two distinct sub-populations based on their level of DCX expression. DCX^hi£rh cells, regardless of location, are restricted to the neuronal lineage or are bone fide neurons, whereas some DCX^low cells retain their multipotentiality. References

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Claims

Claims :

I. A population of DCX^+ve cells enriched for undifferentiated cells comprising cells which are DCX^low.

2. A population as claimed in Claim 1 wherein the cells are PSA^+ve and/or NCAM^+ve.

3. A population as claimed in Claim 1 wherein the cells are β III-tubulin^"ve and/or MAP2^"ve.

4. A population as claimed in Claim 1 wherein the cells are nestin^+ve.

5. A population as claimed in Claim 1 wherein the cells are neurosphere-forming cells.

6. A population as claimed in any one of Claims 1 to 5 derived from embryonic or adult brain tissue.

7. A population as claimed in Claim 6 derived from the SVZ, hippocampus or olfactory bulb.

8. A population as claimed in any one of Claims 1 to

7 including a multipotent stem cell.

9. A population as claimed in any one of claims 1 to

8 comprising undifferentiated cells which are the progeny of said undifferentiated cells.

10. A population as claimed in any one of claims 1 to 8 comprising differentiated cells derived from said undifferentiated cells.

II. A method of generating a population of DCX^+ve cells enriched for undifferentiated cells from a biological sample, comprising the steps of: (i) disrupting the biological sample to provide a mixed population comprising the undifferentiated cells and other cells;

(ii) sorting the mixed population into populations based on the level of expression of doublecortin; and (iii) selecting DCX^low cells to generate said population of DCX^+ve cells enriched for undifferentiated cells.

12. A method for cell replacement therapy in an animal, said method comprising providing a population of cells as claimed in any one of claims 1 to 10 and introducing said population of cells to an animal in need of such treatment.

13. A method as claimed in Claim 12 where the cell replacement therapy comprises the replacement of neural or non-neural tissue.

14. A method for the treatment of neurodegenerative disease in an animal, said method comprising providing a population of cells as claimed in any one of claims 1 to 10, or the progeny thereof, and introducing said population to said animal.

15. A composition for cell replacement therapy and/or for the treatment of a neurodegenerative disease in an animal wherein the composition comprises a population of cells as claimed in any one of claims 1 to 10 and an inert carrier.

16. A method of screening potentially neurologically active therapeutic compounds comprising the steps of:

1) providing a composition comprising a population of cells as claimed in any one of claims 1 to 10 and a medium capable of supporting the growth of the cells;

2) contacting said cell population with at least one candidate pharmaceutical agent; and 3) determining if said candidate agent has an effect on proliferation, differentiation, survival, phenotype or function of said population.

17. A composition comprising a cell population derived from a population of DCX^+ve cells enriched for undifferentiated cells comprising cells which are DCX^low , by induction of differentiation and proliferation, and a medium capable of supporting the growth of the cells.