This work presents the structural and ultrastructural characterization of neurospheres derived from ovarian cortical cells in culture, under conditions previously established.
The identity of spheroid cells as NSCs/NPCs, was confirmed by their molecular signature, that comprised the expression of specific transcripts (nestin, Sox2, Pax6, p75NTR), and by the immunolocalization of the corresponding proteins (nestin, Pax6, p75NTR). A previous study has demonstrated that spheroids generated as described here are neurospheres, by the ability of their cells to self-renew and to differentiate into neurons and glial cells .
The pluripotent transcriptome, Sox2, Oct4 and Nanog, was consistently expressed by sphere cells at all time-points of analysis, which indicated their identity as stem cell spheroids. In addition, results showed a marked increase of Sox2 expression in spheroids sampled on day 10 in culture, in consistency with previous results . Up regulation of Sox2 expression over that of Oct4 and Nanog, is characteristic of pluripotent stem cell specification to the neural lineage , whereby this increase of transcription would accompany this process. In fact, Sox2 is highly expressed in NSCs, and is down-regulated during their differentiation [23, 24], whereby Sox2 is normally considered as a NSC marker. At all time-points of analysis, during the whole culture period, nestin and p75NTR were the transcripts with highest expression. This is consistent with the identity of spheroid cells as NSCs/NPCs, since nestin, a protein that coassembles with vimentin to form class IV intermediate filaments  and participates in mechanisms of filaments disassembly during mitosis , is almost specifically expressed by NSCs, and used as marker for neural precursor cells [27, 28]. As in previous studies , our results showed that 32–53% of spheroid cells showed positive nuclear immunolocalization of nestin. Nuclear localization of nestin, indicates a phosphorylated state of the protein that causes its depolymerization and transport into the nucleus . Even though nestin is most commonly immunolocalized in cytoplasm, its nuclear localization occurs in highly proliferative cells such as in normal NSCs/NPCs during development , in NSCs/NPCs from postnatal CNS , and in cancer stem cells . During neurogenesis, nestin expression is down-regulated, as development proceeds, being replaced by proteins characteristic of neurons and glia . In the current study, a significant percentage of spheroid cells, predominantly placed in the outer sheet cover, show nestin immunolocalization at all time-points of analysis, indicating that NSCs, more immature multipotent cells, highly proliferative, and committed with the neural lineage, are present in these spheroids throughout the whole culture period.
p75NTR was expressed and localized during the entire culture period, as reported previously . P75NTR is a common receptor for all neurotrophins, that partners with tyrosine kinase (TrK) receptors to regulate stem cell differentiation, apoptosis, migration and several other biological processes [34, 35]. p75NTR is particularly enriched in neural crest stem cells , and in neurosphere forming NPCs of diverse origin [37, 38, 39], whereby it is a bona-fide NSC/NPC marker.
Expression of Pax6, a specific transcript of neurogenesis [40, 41], increased on day 15 of culture over that of brachyury. Immunolocalization experiments evidenced that 84–94% of spheroid cells localized Pax6 protein, a NPC specific marker, that promotes NSCs/NPCs differentiation [42, 43], also in neurospheres .
Taken together, gene expression and immunolocalization analyses indicate that most spheroids cells co-localize at least two of the three NSC/NPC markers, in consistency with the previously established identity of these spheroids as neurospheres .
Defining the structural and ultrastructural features of in vitro-derived neurospheres of diverse origin is an essential analytical process for several reasons. First, because it can support the correct evaluation and optimization of cell culture conditions. Second, it can reveal the precise nature of the potentially transplantable cells , since from the analysis of cytoplasmic organelles by TEM, one can foretell graft survival and functionality after cell transplantation or detect oncogenic transformation. Finally, it can demonstrate the reliability of use of neurospheres derived from non-nervous system tissues in eventual cell therapy protocols, once established similar structural and ultrastructural indicators of these cells with respect to CNS derived neurospheres.
Results of structural and ultrastructural analyses, further support the demonstrated identity of these spheroids as neurospheres.
Results of histological analysis, and TEM on semi-thin sections of neurospheres derived from ovarian cortical tissue, revealed that cells were arranged in two well defined compartments: an outer sheet cover, and an inner core area.Three different cell populations were identified by light microscopy: large round cells, intermediate-sized cells, and spindle-shaped cells. TEM analysis helped to elucidate the ultrastructural features of these cells that are consistent with morphological findings described earlier for neurospheres isolated from different tissues and species. Nevertheless, some differences can be appreciated.
The morphological heterogeneity of cells found in neurospheres of this study, has been widely described in neurospheres from central nervous system [9, 10, 11, 12]. Such cellular heterogeneity results, from the diverse topographic distribution of cells within the spheroid, and from the presence of cell subpopulations with distinct survival and proliferative behaviors .
Diverse phenotypes of neurosphere cells have been established by ultrastructural or/and antigenic characterization [9, 10, 11, 12, 16, 17]. Initially, two different cell phenotypes were identified in neurospheres: nestin-negative and nestin-positive cells . Latter studies gave a more complete, but still imprecise, definition of NSCs.
The two main cell types identified by ultrastructural criteria in neurospheres of this study are light cells and dark cells. These cells have been previously described as dark cells, immunopositive for actin, weakly positive for vimentin and nestin-negative; light cells have been characterized as immunopositive for actin, vimentin, and nestin [10, 11]. An earlier study has shown that in these spheroids most nestin-positive cells are found at the spheroid periphery , where there was a major presence of “light cells”, with similar ultrastructural features, than those described by other authors [10, 11], along with abundance of adherens junctions. Results of TEM analyses carried out in the current research show similarities and some discrepancies with previous studies. Neurospheres from ovarian cortical tissue exhibited a core formed by irregularly shaped cells, immature cells, and dark cells, either healthy or degenerating. Most of the cells were arranged around a center, in which dark cells predominated and showed apposition of their cytoplasmic membranes with neighbouring cells, thus forming a compact mass of cells in the center. At the periphery, cells were arranged into epithelium-like layers mostly integrated by healthy mature light cells. These findings show coincidences with previous studies that identify a core of immature cells and an outer part of mature cells [9, 12]. We found a less marked random distribution of cells in the core of the sphere than that reported by other authors [10, 12]. Light cells in the outer part of the neurosphere exhibited two different morphologies: light non-protruding flat cells (LFCs), and light round protruding cells (LPCs). LPCs were shown by histological analyses, and TEM on semi-thin sections, to be leaving the spheroids or aggregating to them. LPCs corresponded to large round cells mentioned previously that were located at the periphery or even outside the spheroid, and were observed by inverted microscopy on days 10 and 15.
LFCs resembled an epithelium, which is supported by two findings: (a) the presence of intercellular junctions, (b) the presence of polarized light cells bearing abundant apical microvilli.
Our TEM results confirm that LFCs of the outer lining of neurospheres established adherens junctions. Adherens junctions have been the only type of cell junction reported in neurospheres, where it might play a role in cell aggregation, spheroid compaction and cell migration by formation of specific cadherin/catenin/cytoskeleton complexes . We found that adherens junctions were located between the apical and the basolateral membranes, serving as a boundary between apical and basal domains . It is known that adherens junctions can be established as temporal cell attachments, to dynamically coordinate intercellular communication . Both characteristics, temporality and high intercellular communication, are landmarks of sphere cells, which would explain the presence of adherens junctions.
In addition, we describe for the first time in neurospheres, the presence of two classes of intercellular contacts between neighbouring LFCs: what we refer to as apical tight interdigitations, and lateral loose interdigitations. Tight interdigitations found at the lateral membrane, were always placed at a very apical position, close to the adherens junction. Loose cellular interdigitations were consistently evidenced over the basal limit of the lateral membrane, beneath the adherens junctions. These types of cell contacts were only found between LFCs of the outer spheroid layer, where they might be participating in intercellular exchanges as can be deduced from the occurrence of multiple laterally expanded cytoplasmic processes, loosely interdigitated with those from adjacent cells through wide intercellular clefts. Such a finding has not been previously described in neurospheres. The only type of cell junction that has been reported is adherens junction, even though intercellular gaps void of cell expansions, and incomplete attachments have been also referred [10, 12]. The presence of the loose cellular interdigitations between LFCs, indicates that they maintain communication, while disengaging from the spheroid is occurring. Subsequently, cells protrude on the neurosphere surface (LPCs), and finally leave the sphere. During histological analyses, we have identified by light microscopy, LPCs on days 10 and 15, either loosely or not attached to the spheroids. In addition, time-course observations under the inverted microscope during spheroid culture, revealed the presence of a large number of round cells that arised from spheroids and migrated towards particular areas of the growth surface. Migrating round cells frequently showed filopodia and eventually, morphological features of neural cells. An alternative possibility is that, at least part of these round migrating cells, would be aggregating to a pre-existing spheroid. Our TEM results, add support to the first possibility, that will be addressed in future experiments. In any case, both migration followed by morphological differentiation, and migration followed by cell aggregation to generate a new spheroid most probably occur simultaneously, since prior studies have demonstrated that these spheroids self-renew, and their integrating cells are able to differentiate into neurons and glia . Such cell disengaging at the lateral membranes seems to begin in the basal domain and to move towards the apical cell border, where adherens junctions and focal tight intedigitations are present, until the cell is finally released.
As we have already mentioned, LFCs placed at the outer spheroid layer exhibited signs of apical-basal polarity, such as the presence of apical microvilli and adherens junctions. Intercellular junctions play an essential role in maintaining apical-basal cell polarity since they serve as a physical limit between the apical/luminal and basolateral/luminal cell compartments . Early cell-cell contacts during spheroid formation would induce the formation of intercellular junctions, which recruit and activate polarity proteins. In turn, polarity proteins regulate further maturation of adherens and tight junctions.
However, some observations might indicate that loss of polarization in LFCs could be underway on 15 days of culture. First, as we have already described the presence of loose interdigitations between neighbouring cells, seems to be reflecting cellular disengagement at this time (LPCs), which is a previous step for the cell to exit the sphere; loss of apical-basal polarity appears to be an essential requirement for cell disengaging, and is related with its proliferative activity and multipotency , being a characteristic feature of normal migrating cells, such as neural crest stem cells. Loss of apical-basal polarity is also characteristic of cells involved in tumor formation and metastasis [49, 50, 51, 52]. Second, we have shown that intermediate filaments were very abundant in light cells of neurospheres, particularly on day 15 of culture, when they were either strikingly spreaded out through the cytoplasm, in a non-polarized way, or arranged in bundles, as reported by Lobo et al. . In other studies, intermediate filaments are mentioned as occasional findings . In epithelial barriers, intermediate filaments provide mechanical strength and cellular shaping, and participate in maintenance and cross-talk with tight junction complexes [47, 53, 54, 55]. Intermediate filaments create polarized scaffolds to preserve epithelial asymmetry and cell shape . As an example of such function, in ependymal cells, intermediate filaments together with apical F-actin bundles, maintain the structural integrity of the central canal . In epithelial cells, intermediate filaments localize in apical regions, whereas in non-polarized multilayered epithelia their localization is ubiquitous. In fact, in non-polarized cells intermediate filaments are dynamic and mobile . Even though the presence of filament bundles in the cytoplasm of light cells of neurospheres has been reported , their functional significance has not been yet explained. Our TEM results, along with observations under the inverted microscope, support the view that, since cell polarization in culture occurs slowly during several days, intermediate filaments might contribute to maintain structural integrity of spheroids for days.
Our results regarding arrangement of intermediate filaments in light cells of neurospheres, support the hypothesis that by day 15 of culture, loss of structural integrity and polarity of LFCs might be initiating, before LPCs leave the neurosphere. Therefore, we hypothesize that LFCs might become LPCs, and that progressive loss of apical-basal polarity would finally result in cell rounding prior to cell disengaging from the spheroid.
The abundance of intermediate filaments in light cells is supported by a previous study  in which most nestin (an intermediate filament protein) positive cells were more frequently found at the spheroid periphery, where light cells are more abundant.
Light cells placed at the outer sheet of the neurospheres (LFCs and LPCs), beared abundant microvilli at their apical membrane. The presence of apical microvilli is a characteristic feature of embryonic stem cells [58, 59, 60], epithelial cells of embryoid bodies , and neuroepithelial and neural progenitor cells during neurogenesis , but it has been reported to be a rare finding in neurospheres .
Apical microvilli found in light cells of the outer sheet cover from the current study, resembles those of ependymal cells. Microvilli of ependymocytes, considered as the source of multipotent adult NSCs [10, 63], provide the capability of adaptation of cells to different external conditions . It is possible that microvilli of outer sheet cover cells of the spheroid have a similar function, that is to become organized and positioned against external forces.
Cell rounding and the presence of microvilli, as shown in cells leaving the spheroids or aggregating to them, is a characteristic feature of progenitor cells that initiate migration with no adhesion to a growth surface . Microvilli also function in absorption and secretion of molecules. The high metabolic activity of the light cells, evidenced by the abundance of cytoplasmic organelles, phagosomes and lysosome-like structures, correspond to the intense biochemical exchanges at the periphery of the sphere, as previously have been noted [9, 12], where the microvilli protrude.
Taken together, our results of TEM analysis of light cells of the outer sheet of the neurosphere, regarding polarity, intercellular junctions, and apical microvilli, seem to resemble that of epithelial cells of choroid plexus in the brain. These cells are the primary producers of the cerebrospinal fluid (CSF) and are responsible for establishing the blood–CSF barrier . These cells display a characteristic polarity with microvilli, cilia and tight junctions at their apical side, and adherens junctions, gap junctions and desmosomes or basal infoldings at the basolateral side [66, 67, 68].
Within both dark and light cell populations, different phenotypes were identified: healthy dark cells, degenerating dark cells, flat light cells, protruding light migrating cells. Rather than subpopulations of cells , these cells might be transitional states from one or two original cell types that differentiated in culture within the spheroid.
Structural and ultrastructural results revealed that the outer layer comprises cells with signs of high metabolic activity, whereas the core of the neurosphere is the site of necrotic, apoptotic and phagocytic phenomena. These results are consistent with previous investigations showing high cell heterogeneity in the core, where apoptosis, necrosis and autophagy are frequent events, and the outer layer comprises healthier cells with intense protein synthesis and high mitosis rates [9, 12]. The compartimentalization of these phenomena may be related with the different capacity of the cells to reach culture medium nutrients and oxygen in the outer sheet or in the inner core of spheroids.
The increased incidence of degenerating cells, apoptotic phenomena, and phagocytic activity in the spheroid core, suggests that mechanisms for selection and elimination of overproduced cells are operating as autophagy-regulated phagocytosis  in this part of the spheroid. These mechanisms, known to function during development, contribute to eliminate apoptotic bodies during embryo differentiation .