In this study, we describe the unique properties of a putative in vitro NSC niche. We previously defined a set of criteria that an in vitro system should satisfy to appropriately model elements of the developing and adult subventricular in vivo NSC niche . These properties include the presence of NSCs; production of signaling molecules characteristic of NSC niches; the presence of transit-amplifying cells; the ability to produce both neurons and glia; the presence of a basal lamina and extracellular matrix; the contribution of radial glia as the primitive source of stem cells and as a scaffold for organization of the niche microenvironment; and autonomous production of cellular and molecular factors necessary for self-renewal and differentiation of resident stem cells. Because our mature culture system exhibits all of these characteristics, we propose that it is an in vitro NSC niche.
Our electron micrographs show the complexity of the aggregates and the strands of many cable-like processes between them (Fig. 1A-F). Based on our electron microscopy, we conclude that the in vitro NSC niche is a complex network of interconnected aggregates, each of which contains presumptive stem cells capable of differentiation. This observation supports our earlier findings that NSCs and their progeny are maintained within the in vitro NSC niche .
Although there is considerable cellular heterogeneity in the in vitro NSC niche, the cultures do retain a consistent molecular signature. First, the initial retinoic acid induction  causes the niche to become predominantly neural, consisting of a diversity of cells from NSCs to mature neurons, glia, and oligodendrocytes (Figs. 2, 6 and 8) . However, there is continued evidence of non-ectodermal lineages present in the cultures (Fig. 6E). Though there is a steady increase in expression of genes indicative of mature neural cells between Days 10 and 14 (Fig. 6A and 6C), there remains a persistent presence of radial glia, NSCs and transit-amplifying cells in the cultures (Figs. 2, 6 and 8). A clear progression of expression of Pax6 through TBR2 to TBR1+neurons is indicative of neurogenesis in the SVZ [22, 23]. The presence of Pax6 and TBR2 expression may suggest this progression in our cultures. However, there is not a clear and significant increase or decrease in these transcripts during culturing.
The development of the in vivo subventricular NSC niche is dependent on a network of ECM components [17, 18, 53] and their receptors [3, 4, 10, 17]. Likewise, cells of the in vitro NSC niche express the specific ECM components suggestive of stem cell maintenance including the gamma-1 subunit of laminin, nidogen 1, collagen IV, perlecan, and syndecan-3 (Fig. 2C, Fig. 7A and 7C). Expression of E, P, VE, and N-cadherins suggests that there are cells within the in vitro NSC niche representing various levels of differentiation and lineage commitment, and may further suggest a mechanism for the organization of the aggregates based on cadherin expression. It is possible that the level of lineage commitment of neural progeny determines the probability of their association with different locations in the niche . β-1 integrin acts to regulate stem cell identity and anchor the putative NSC to the ECM [10, 17, 52]. β-1 integrin expression in the in vitro NSC niche suggests that it may retain these roles in our cultures (Fig. 2C, Fig. 7C). Wnt1 and Bmp4 expression (Fig. 7G) are likely involved in additional stem cell regulation, having been implicated in self-renewal pathways [1, 54].
The maintenance of a NSC population within the in vitro NSC niche is strongly supported by our protein expression data (Fig. 8). There is a NSC population that co-expresses PDGFRα, GFAP and CD133, possibly indicating a transition from embryonic NSC to adult NSC in neural cultures [6, 51]. Cells that express CD133 but not PDGFRα or GFAP may be indicative of early embryonic NSCs, adult ependymal cells, or from a non-neural lineage. High expression levels of VE-cadherin on Day 14 may indicate that some CD133+ cells are of a hematopoietic lineage . Interaction with cells of a hematopoietic lineage within our cultures could be necessary for NSC and niche maintenance [12–14].
Development of the in vitro NSC niche appears to be more dependent on density (Additional file 2), than exogenous ECM application (Fig. 3 and Additional file 3). The cues for aggregation of the cells are not known, but β-1 integrin and other ECM components and receptors likely contribute to the aggregate formation, process extension, and cellular migration within the developing niche cultures [9, 20, 55].
Apoptosis occurs concurrently with the development and reorganization of the in vitro NSC niche, and a role for apoptosis has been demonstrated in this development in vivo [24, 56]. Our data suggest that primarily the mature cells of the in vitro niche exhibit apoptotic activity (Fig. 5). This may be due to metabolic stress induced by serum deprivation, or may relate more directly to the presence or absence of downstream neural connectivity . Varicosities on processes and cytoplasmic blebbing (Fig. 1E) also suggest a role of apoptosis in maturation of intercellular networks and remodeling of the niche cultures [24, 57].
Here we have described the morphological properties of the in vitro NSC niche, the molecular components produced by cells of the niche cultures, the maintenance of a NSC population in the niche and the persistence of neurogenesis. We previously described use of the 4-/4+ retinoic acid neuralization protocol  in preparation of neuralized ES cells for transplant into mouse models of neurodegeneration [35, 58]. Meyer et al.  described few Nestin-expressing neural precursors present when neuralized ES cells are dissociated and plated at low densities for 8 days as adherent cultures. Retinoic acid induction promotes neural differentiation of ES cells and additional time in culture favored differentiation of post-mitotic cells [9, 34, 35].
The protocol used to produce the in vitro NSC niche enriches for NSCs while retaining the potential for further neurogenesis (Fig. 8), and neurogenic potential appears to be increased significantly when compared to retinoic acid induction alone (Fig. 6B and 6D). Smith and colleagues have described a method of ES cell neuralization that results in a very homogeneous culture of NSCs . With regard to transgenic modifications of a stem cell culture, homogenous populations may more easily be transduced with an expression vector of interest . In cell culture, it is possible to direct neural fate specification through substrate selection , but it may be difficult to maintain broad potential for differentiation from a homogeneous pool of precursors .
There are complex molecular and cellular interactions that regulate NSCs in the in vivo NSC niche [1, 3, 4]. Initial efforts in isolation and passage of primary NSCs from developing mammalian brain were achieved using neurospheres, floating clusters including NSCs, neurons, and glia . NSCs within neurospheres retain competence for self-renewal and are multipotent , but the cell-types present within neurospheres may change with repeated passage . If neurospheres are allowed to adhere in close proximity they exhibit unique properties, such as migration of cells between clusters along complex processes. ffrench-Constant and colleagues suggested that a culture of adherent neurospheres with cells migrating between them was similar to SVZ explants . Likewise, there are similarities in morphology, cell-types, and cellular migration in our ES-derived in vitro NSC niche, neurospheres in adherent cultures, and the SVZ NSC niche .
The in vitro NSC niche is the first neurogenic culture system to be produced without the application of exogenous mitogenic factors and complex physical scaffolding. Although the roles in niche formation of endogenous factors that influence the derivation and maintenance of this the in vitro NSC niche microenvironment have yet to be tested, our model is able to recapitulate many characteristics of the previously described in vivo NSC niche. Therefore, the in vitro NSC niche may be a valuable tool for assaying interactions within the in vivo SVZ or SEZ niche.
Exogenous application of ECM and the 3-dimensional nature of aggregates in the in vitro NSC niche provide depth to the culture system that appear to be required for growth factor production, cell migration [9, 64] and stem cell maintenance (Fig. 8D). We addressed whether 3-D scaffolding might give additional benefit in bringing the in vitro NSC niche more fully into the third dimension [65–68]. To that end, we chose to use PuraMatrix hydrogel scaffolding system. First, PuraMatrix hydrogel is comprised entirely of polymeric amino acids and does not require serum or any components of serum. Our system forms in the absence of mitogens from Day 8-14. Any serum component added with a matrix could result in differentiation of the resident NSCs prior to niche formation. Second, the concentration of the RADA-16 component of PuraMatrix can be adjusted to be permissive to neurite outgrowth . Third, PuraMatrix "self-assembles" and the use of a defined component, consisting of only amino acids, provides structure without unknown components. Finally, the ability to add binding sites specific to the promotion self-renewal or differentiation  offers the possibility of deriving a unique scaffold optimized for niche growth.
We have been able to reproduce niche morphology of the 2-D system in 3-D PuraMatrix when a 10-fold increase in ECL concentration is used (Fig. 3), but the pattern of mRNA expression by the niche changed in PuraMatrix (Fig. 4). Both 2- and 3-D cultures were capable of integration and survival in organotypic slice cultures, suggesting they are a possible source for cells in transplant studies. Further work is required to optimize the 3-D culture methods and characterize the cellular outcomes before a 3-D NSC niche can be widely used for transplantation.
The in vitro NSC niche may provide a novel source of cells for therapeutics. Because this culture system includes CD133+/PDGFRα-/GFAP- NSCs with properties in common with embryonic NSCs  along with CD133-/PDGFRα+/GFAP+ NSCs similar to those of the SEZ  (as well as a possible transitioning population of CD133+/PDGFRα+/GFAP+ cells), it may have a broader potential for therapeutic value than any single cell population (Fig. 8D). It will likely provide a source of cells for transplant able to adhere to the ECM expressed by recipients of a wider range of ages. Furthermore, as the in vitro NSC niche is derived from an ES cell population, transgenic modification can be accomplished followed by clonal expansion .