This work is an attempt to purify immature neuronal cells from a renewable source of NSC progeny based on their size and internal complexity. Our findings show that immature neurons have very small cell bodies and nuclei, and are distinguishable from astrocytes using microscopy ( and Fig. S1A
–C). Exploiting these characteristics via flow cytometry, where distinct cell populations can be separated based on light scatter properties, we obtained an enriched immature neuronal population that developed a GABAergic phenotype and was minimally contaminated with glial cells. Further enrichment, to near homogeneity (97%), was achieved based on a negative (eliminate O4+ & A2B5+ cells) or positive (PSA-NCAM+
) selection. These immature neurons are sensitive to their environmental conditions and are not very robust, however, their survival and maturation, both in vitro
and in vivo
, could be enhanced by exposure to BMP4.
Neuronal cells have been enriched from primary brain tissue based on cell size 
, transgene 
and PSA-NCAM expression 
. While these approaches provide enriched populations of cells for pre-clinical studies, they are limited in their clinical application due to supply and ethical issues of harvesting donor tissue from primary human cells. ES cells represent a viable alternative, and flow cytometry has been used together with transgene expression to derive purified neuronal populations with attenuated tumor forming ability 
. This has also been combined with immunopanning for PSA-NCAM to further enrich the neuronal population 
. Although potentially useful as a renewable source of cells, genetic alteration of donor cells is less than ideal and ES cells carry an inherent risk of tumor formation that may be unacceptable for wide clinical application. More recently, induced Pluripotent Stem Cells (iPS) 
have been used to generate neural cells 
that may serve as a more ethically acceptable autologous donor population, in particular when induced through non-genetic methods via a transient neural stem cell population 
One of the risks in using any renewable precursor population as a donor source of cells is the potential for uncontrolled or unwanted proliferation. Recently, it has been proposed that NSC progeny are the in situ
source of gliomas 
and that extensive culture of adult stem cells may generate malignant phenotypes 
While this is countered by a detailed study of extensively passed adult NSCs demonstrating their genetic stability, resilience to transformation and inability to form tumors in immunocompromised mice 
, a report by Amariglio and colleagues of a pediatric patient who had received repeated intracerebellar and intrathecal injections of large numbers of primary fetal CNS cells, and presented four years later with a well differentiated benign glioneuronal neoplasm, suggests a more cautious approach. While the tumor was likely the result of the large number of neural progenitor cells 
, the presence of NSCs within the slurry of donor cells and the resultant undesired growth (although not transformed nor malignant) raises concern and the need for more defined populations of cells for transplantation. Our sorted P1 population was not only highly enriched with immature neuronal cells but also contained a precursor population (include stem cells and progenitor cells) of approximately 1
1111 cells, which contrasts the 1
83 precursor cell frequency in the overall population, representing a 13 fold reduction. More importantly though was the change in the bona fide stem cell population (based on the N-CFCA 
) where its frequency was reduced from 1
3114 to 1
200,000, a 64-fold change. While we were unable to eliminate all of the stem or precursor cells, the reduction may be functionally important. To test this we implanted 100,000 cells from our P1 population (derived from a tau-GFP transgenic) into NOD/SCID animals and 4-weeks later counted the number of DAPI+/GFP expressing cells that were immunoreactive for Ki-67 or MCM2. A very small population of cells was found to express Ki-67 or MCM2 (0.21±0.12% and 0.29±0.17%, respectively), revealing that our sorting procedure did not eliminate all cells witha proliferative potential. This contrasted the results of Yan et al.
who reported that 3–5% of their NSC progeny expressed Ki-67, 3–6 months post-implantation. While a small fraction of cells retained the ability to proliferate we observed no clusters of proliferation, proliferative rosettes or masses of cells that would indicate delirious uncontrolled growth, rather the few cells we noted (1 out of 400) were randomly scattered around the remaining surviving cells. It is important to note that these data were generated using only the P1 population (FSClow
) without additional negative or positive selection and a further reduction in the number of dividing cells may be achieved by employing these additional purification strategies.
Our findings demonstrated that addition of BMP4 (at 20 ng/ml) to our sorted immature neurons increases their survival and is consistent with the report by Lim and colleagues 
who reported that BMP4 increased the survival of purified immature neuroblasts from dissociated SVZ cultures. Furthermore, it has been shown that in the olfactory bulb, high concentration of BMP4 inhibits proliferation of neuronal progenitors, promotes their exit from the cell cycle and initiates differentiation. In addition, BMP4 promotes glial differentiation of progenitor cells from the forebrain subventricular zone 
and CNS derived tumor cells 
Treatment of sorted P1 population with BMP4 did not increase the number of GFAP IR cells after six days of culture (data not shown), nor did we observe any GFAP positive cells 4 weeks after transplantation into the adult striatum of BMP4 treated P1 cells. This suggests the P1 cells are committed to neuronal fate and that BMP4 enhances their maturation and survival as demonstrated by an increase in the percentage of NeuN IR cells in BMP4 treated transplanted P1 cells, relative to controls. Our electrophysiological data support the role of BMP-4 in supporting functional maturation (showing action potential from RMP or establishment of Na+ channels in cell membrane).
Application of this methodology for purification of a defined population of highly enriched immature neuronal cells from a heterogeneous population of human NSC progeny provides a renewable source of cells that may have potential for in vitro
studies such as drug screening, neurotoxicolgy, electrophysiology and to act as a source of cells for implantation to repair the injured CNS. However, additional work still needs to be carried out to determine if the purified implanted donor cells are able to functionally integrate. There are many research studies showing therapeutic application of neural stem and progenitor cells in animal models of diseases including reduction in seizure activity 
, alleviating neuropathic pain and restoring disrupted neuronal circuitry in spinal cord injury 
, increasing axonal sprouting in neonatal hypoxic ischemic brain injury 
, and delaying onset and progression of amyotrophic lateral sclerosis (ALS) 
. Applying the methodology developed in this study to human neural stem cells, we hope to be able to define the starting population of donor cells (e.g. 60% neurons, 40% glia or 90% neurons and 10% glia) which may provide the opportunity to determine the cell type(s) responsible for a phenotypic change, to investigate the potential mechanisms, and adjust dosing strategies based on a particular disease or disease stage. Application of supportive factors that increase survival of the immature neurons after transplantation, in particularly when used together with a permissive vehicle such as a 3-dimensional matrix, may increase the practical application of NSC as a source of donor material for cell replacement therapies.