Introduction

The stem cells have a high capacity for proliferation and self-renewal, differentiation in the main lineages of their original tissue and tissue and/or organ regeneration. Neural stem cells (NSCs) are proliferative cells that give rise to neurons and glial cells and are present in the embryonic, neonatal brain and in some regions of the adult central nervous system (CNS). The use of NSCs to promote the repair of brain injuries has proved to be an efficient and promising approach in neurology field [1, 2]. Since the first publication in 1992 [3], many research groups have investigated the mechanisms of different neurodegenerative diseases using in vitro models, especially neurospheres. In this review, we describe the principles of neurosphere assay and analyze the different aspects of research with neural stem cells, highlighting the main implications of an in vitro model that can help explain the outcomes of some neurodegenerative diseases.

Neurodevelopment

During the development of the CNS of mammals, neurons and neuroglial cells (astrocytes and oligodendrocytes) are generated from multipotent cells, with neurons being the first cells generated in the process, mainly during the embryonic period. Historically, neurogenesis was believed to cease at the end of embryonic development. However, today we know that this process is continuous throughout life and occurs in different regions of the adult brain [4].

The understanding of adult neurogenesis has progressed significantly and we already have great knowledge about the biology of this phenomenon, from the location, proliferation and specification of the fate of neural stem cells migration, neuronal maturation and synaptic integration of newborn neurons [5, 6]. Neural stem cells per se generate distinct cell types, the progenitors. These are proliferative cells, but they originate cells more restricted to the lineage and these neuronal and glial progenitors cannot self-renew and express some lineage marker gene transcripts. With this, we can efficiently analyze these new cells through the isolation and in vitro analysis of the progenitors derived from the adult CNS, opening possibilities for studies in the repair of the CNS after injuries, using cell replacement therapy in the repair of degenerative diseases [7, 8].

In recent decades, the development of refined techniques has resulted in a lot of new research showing that neurogenesis, the birth of new neurons, occurs in limited and specific regions of the adult brain, with significant numbers of neurogenic cells. In mammals, the process of neurogenesis occurs in brain regions called “niches”, where NSCs are present. These regions include the subgranular zone of the hippocampal dentate gyrus (SGZ), subventricular zone of the lateral ventricles (SVZ), external germ layer of the cerebellum (EGL), subcallosal zone (SCZ), corpus callusum (CC), among others. However, the regions most frequently used by researchers to study the neurogenesis and isolation of NSCs are mainly the SVZ and SGZ of the hippocampal dentate gyrus, which we will report throughout this review [9, 10].

Both regions are responsible for maintaining neural precursor cells and generating new neurons. However, on the side walls of the lateral ventricles is the largest germinative zone of the brain of adult mammals. The SVZ is the region responsible for highest production of neuronal cells in the embryonic and adult phases, generating highly proliferative progenitors that can be used for neuroregenerative therapy [9, 11].

Neural precursor cells can actively participate in the repair process and intrinsically can synthesize many molecules useful for tissue regeneration. They constitute an extremely diverse population of cells, with specific morphology and markers of their germinal regions, exhibiting different characteristics and functions depending on their proliferative state and region. They were primarily isolated from the CNS of rodents by means of cell culture and these cells can be expanded and modified, maintaining their multipotentiality in many passages. Because it is easy to obtain, the manipulation of NSCs allows us to accurately analyze the intrinsic and extrinsic mechanisms that are involved in neurogenesis [3, 12, 13].

The existence of a type of multipotential stem cell in the CNS supports the findings that precursor cells derived from different regions of the developing and adult brain have similar properties. Adult neurogenesis in the hippocampus and olfactory bulb is an extremely dynamic process. Its regulation occurs through different points of stimulation and the understanding by which mechanisms this phenomenon happens can significantly enrich our knowledge about the etiology and pathophysiology of some neurological diseases [14, 15].

The neurogenic process needs precise control, since any physiological change can interrupt the process, causing apoptosis of the neuroprogenitors and deregulation related to hippocampal hypoplasia and neurodegeneration. In Parkinson's disease (PD), for example, the depletion of dopaminergic neuronal circuits is associated with a neurogenic deficiency observed both in the SVZ and in the SGZ of the dentate gyrus. However, the increase in neurogenesis is not necessarily beneficial and may be associated with other pathologies [16, 17]. Some studies suggest that exacerbated neurogenesis, in response to brain damage, is related to the development of epilepsy and its reduction may be involved in the pathophysiology of psychiatric disorders and neurodegeneration disease [18,19,20].

Studies show that NSCs respond to different stimuli during embryonic and adult development, especially those related to proliferation, such as growth factors. In vitro, these cells renew themselves by signaling, dividing and migrating from the matrix cell cluster—called spheres—generating new cells, such as neurons and glial cells, in addition to new NSCs that will form new matrix spheres after mechanical dissociation of in vitro-grown spheres. The spheres generated contain the same undifferentiated phenotype as the original sphere, thus demonstrating that NSCs have a high potential for self-renewal. Due to these characteristics, these cells are a promising tool for the study of neurodevelopment, as well as for identifying the etiology of different neurodegenerative diseases [15, 21].

The mechanisms that control neurogenesis are not yet fully elucidated, especially in the postnatal period, but it is known that changes in its process are involved in different pathologies. For this reason, therapeutic strategies focused on the neurogenic process need to be tested in preclinical and clinical trials.

Neurospheres

The SVZ and SGZ of the hippocampus are neurogenic regions in the adult brain that contain multipotent cells that renew and differentiate into all types of neural cells. The NSCs generated in these regions of adult mammalian brain play an important role in replacing post-mitotic cells and, consequently, in regenerative repair after injury [22, 23].

To isolate and expand NSCs, a culture system known as the neurosphere assay (NSA) is performed in order to generate primary cells capable of differentiating into the three main cell types of the CNS [24]. In vitro neurospheres were demonstrated to derive each from one neural stem cell and to consist in a clone of progenies including neural progenitors and new stem cells [25]. Neurosphere-constituting cells are non-adherent. Mature spheres display variable sizes (between 100 and 200 µm) and, once deprived of the mitogenic growth factors (EGF, bFGF), adhere to the substrate and display cell differentiation into neurons, astrocytes and oligodendrocytes [3, 25, 26].

The NSCs and patterns within the spheres may have specific regional and temporal characteristics in relation to growth, differentiation and specific gene expression in the region. But, regardless of regional origin, all neurospheres contain cells of different subtypes and are able to maintain the molecular patterns of expression of specific region genes across in vitro passages [27, 28].

Neurospheres generated from the different regions of the CNS express unique markers for each region, indicating that the original cells were in fact specified regionally. Since its discovery, new sources of neurosphere-forming cells have been investigated and different culture protocols have been developed to maintain, expand and differentiate them, thus allowing the characterization of these neural progenitors soon after isolation [29, 30].

Neurosphere culture is an excellent tool for obtaining and expanding neural and progenitor stem cells, in addition to allowing to analyze their properties under controlled conditions. Due to their high proliferation capacity, neurospheres can be dissociated and cultivated in many passages [31]. A large number of cells derived from SVZ and SGZ can be obtained after isolation and expansion, when in media enriched with growth factors. After being differentiated into a neuronal lineage, the cells have great potential for studies in the field of neurosciences such as to test new drugs, assist in clarifying and preventing the progression of neurodegenerative diseases and even elucidating the processes involved in embryogenesis [11, 32].

The proliferation of NSCs continues on the side walls of the lateral ventricle in the brain in the postnatal and adult periods. All neurosphere cultivation methods previously reported by researchers have been optimized for working with NSCs isolated from rodents of different ages. However, neonatal SVZ is more dense and matures from birth to mature organization, around 15 postnatal days [30, 33].

The most important characteristics that the cell model using neurospheres can offer are (1) the identification of neural stem cells; (2) the simplicity of the technique; (3) serve as a starting point for studies of molecular and biochemical mechanisms of neurodevelopment and (4) a means of tracking factors that may trigger changes in neurogenesis and trigger CNS diseases.

Neurosphere assay

In general, in order to obtain neural stem cells and neural progenitors in culture, and generate the neurospheres, it is necessary to follow 5 fundamental steps: obtaining the tissue, dissociation, cell isolation, cultivation and maintenance (Fig. 1). Firstly, it is necessary to extract the nervous tissue, in this case the brain, and secondly the region of interest, which may be SVZ or SGZ. Then, the tissue needs to be dissociated enzymatically using enzymes such as trypsin/EDTA, papain, accutase and collagenase, followed by mechanical dissociation [11, 34, 35]. The cell pellet generated is cultivated on non-adherent substrate using a serum-free growth medium with mitogenic growth factors in a humidified incubator at 37 °C and 5% CO2 [11]. The cells of the seeded tissue start to form neurospheres from the tenth day and they can be dissociated for sequential passages, only with the change of the growth medium [36]. The generated neurospheres, being dissociated in multiple passages or not, remain in suspension of single cells until they are cultured in adherent plates coated with poly-l-lysine and finally, when removing growth factors from the medium, adhesion to the substrate and differentiation into neurons, astrocytes and oligodendroglia occurs [27, 35,36,37,38,39].

Fig. 1
figure 1

Five fundamental steps for neurosphere assay from mouse cerebral tissue. The generated neurospheres, being dissociated in multiple passages or not, remain in suspension of single cells until they are cultured in adherent plates coated with poly-l-lysine and finally, when removing growth factors from the medium, adhesion to the substrate and differentiation into neurons, astrocytes and oligodendroglia occurs

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The cultures generated from the SVZ and SGZ regions have similar morphological characteristics. However, SVZ-derived cells proliferate more quickly, forming larger neurospheres, with a much larger number of neural precursor cells residing in SVZ compared to SGZ [38]. From these and other evidences, the SVZ became the most isolated region for the realization of the NSA.

Culture medium and maintenance

The growth medium is basically composed by DMEM/F12—Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12 supplemented with 100 U/ml penicillin/streptomycin, 2 mM GlutaMAX, 20 ng/ml basic fibroblast growth factor (bFGF) and 20 ng/ml epidermal growth factor (EGF), 2% B-27 and 1% N2 supplement, the latter is not mandatory [11, 36, 39, 40].

The neurospheres culture is non-adherent, so to maintain this pattern, these cells are grown in the absence of serum and in untreated plates, because components of serum and the adhesion treatment of conventional plates favor the differentiation of NSCs and neural progenitors from irreversible way [11]. It is known that supplementation with serum promotes growth and stimulates the proliferation of cells in culture. In this cell culture model, these processes are promoted through supplementation with B-27/N2, compounds that act on cell growth, viability and survival of embryonic, post-natal and adults nerve cells of the hippocampus [39, 41].

To keep the neurospheres undifferentiated, it is necessary to supplement the medium with growth factors, especially EGF and FGF. In neurospheres, EGF increases the proliferation and survival of NSCs and neural progenitors, in addition to keeping them undifferentiated, while FGF activates neurogenesis and self-renewal [42]. Therefore, it is recommended that these growth factors be combined in the neurospheres growth medium and added to cells every 3 days to maintain undifferentiated status [11, 43].

In addition to these supplements, the culture of neurospheres requires the addition of micronutrients such as L-glutamine. This amino acid is an energy source for cells, being present in different culture media. Cells that divide rapidly, such as neurospheres, prefer media rich in L-glutamine, as these cells have a high rate of proliferation, need to rapidly synthesize proteins and nucleic acids and L-glutamine assists in these processes. However, after metabolization this compound is converted into ammonia, being toxic to cells, so the culture medium needs to be constantly renewed. While neurospheres are being formed, the basal environment is not renewed, it is only supplemented with mitogenic factors. So that L-glutamine does not hinder the development of neurospheres, this culture is supplemented with GlutaMAX, a stable component in an aqueous solution that is not spontaneously degraded and provides energy to the cells as well [11, 38].

All of these components must be added to the DMEM/F-12 basal medium, a versatile medium with high concentrations of glucose, amino acids and vitamins, ideal for the growth of neurospheres [11].

Morphology and phenotypic characterization

When we look at neurospheres in a phase-contrast microscope, it is possible to observe shiny spheres, with varying sizes (between 100–200 µm), composed of hundreds to thousands of cells. Generally, they have a clear and rounded nucleus with dispersed chromatin and two nucleolus. Neurospheres have morphological diversity, where larger spheres (that exceed 200 µm) generally have a darker core, with denser cells inside [44,45,46]. This indicates that in this region there is a high rate of apoptosis, because the larger the spheres the less nutrients reach the cells located in its center.

Regarding their phenotypic characterization, neurospheres are subjected to analysis of molecular markers and gene expression of each cell population. The NSCs do not have a specific molecular marker described so far, which makes it difficult to characterize [11]. However, these cells can transiently express the intermediate filament protein, nestin. This protein is found in nerve cells and can be used as a marker of neural precursors. In addition to NSCs and neural precursors, neurospheres have other cell types inside, which have molecular markers, which are: (1) type B cells or astrocytes expressing glial fibrillary acidic protein (GFAP), high-affinity glutamate/aspartate transporter (GLAST) and CD133; (2) type C cells expressing SRY-box transcription factor-2 (Sox2) and oligodendrocyte transcription factor (Olig2); (3) type A cells what expressing III beta-tubulin (Tuj1). Neurospheres are also positive for the cell cycle marker Ki-67, indicating that these cells have a high proliferation rate [11, 47, 48]. Thus, the presence of these different markers in the neurospheres indicates that this cell population is naturally heterogeneous and composed of cells in different stages of differentiation such as stem cells, proliferating neural progenitors and post-mitotic neurons and glia [41, 47, 48].

Advantages and disadvantages

The NSA assay was the first in vitro model able to demonstrate that the adult brain has effective neurogenic regions composed of a large number of neural stem cells, making it an important tool to evaluate proliferation, self-renewal and multipotency of stem cells and neural progenitors. The neurosphere culture is an excellent model to study neurogenesis, neuronal development and neuroregeneration, because they are easy to manipulate and respond well to extrinsic stimuli present in your microenvironment [41]. Unlike most primary cultures of CNS, neurospheres are relatively easy to grow, subject to multiple passages and able to differentiate into neurons and glial cells, making it an essential tool for studying aspects of the developing brain and CNS pathologies that need effective medical treatment [11, 49].

However, due to its heterogeneous character and variations in methodology, the NSA should be used with great caution. A disadvantage of most protocols for this type of culture has been the need to use a relatively large number of animals, because the yield of cell isolation is generally low [38] and this variation in cell density can generate changes in the microenvironment, affecting the proliferation. In addition, the concentrations of mitogenic factors in the culture medium, dissociation technique and number of passages are factors that can alter the composition and properties of neurospheres, interfering mainly in their neurogenic potential [41, 50, 51]. Another disadvantage in this culture system is that the neurospheres, when transplanted into the brain for therapeutic purposes, mostly differentiate into glial cells, generating few neurons [41]. Therefore, this technique becomes more useful in the regenerative medicine of glial and non-neuronal diseases.

The combination of the progressive loss of neurogenic potential after several passages with the low neuron yield reveals the need for refinement of this culture system, which supports greater expansion of stem cells and neural progenitors, increasing their capacity for neuronal differentiation. Thus, with a very robust system, we can expand our knowledge about NSCs and their therapeutic applications.

Current research scenario

The isolation of CNS neural precursors, from embryonic and adult tissue, through the neurosphere formation assay was first described in 1992 by Reynolds and collaborators and since then the application of culture protocols for the isolation of NSCs has already been seen as a promising strategy for a better understanding of the behavior of different types of NSCs [3, 52].

Since these cells were first described in the brain of mice, the use of fetal brain tissue in both, cell therapy and transplants fields has received a lot of attention. Expectations and great interest were generated in the possibility of NSCs becoming an opportunity for treatment of neurodegenerative disorders [53, 54]. The biggest problem yet to be solved is the fact of how to direct and control the differentiation of specific cell phenotypes necessary for the replacement and repair in each disease.

Neurospheres offer an excellent source of neuroprogenitors for the study of neuronal development and differentiation. These multicellular spheres are capable of reproducing functions characteristic of the developing brain, such as proliferation, migration and differentiation. In neurosciences, the application of stem cells and neuroprogenitors has been studied with a focus on the treatment of neurodegenerative diseases such as sclerosis, stroke, cancer and trauma in order to recover damaged tissues [55].

In Table 1 we compile the main studies related to neurological disorders, including Alzheimer, Parkinson, demyelinating diseases, epilepsy and glioma associated with degeneration, which used the neurosphere assay as an investigation tool. Several of these studies have used human fetal brain tissue, human forebrain and rodent brain to isolate and cultured neural progenitors, as neurospheres, and apply them as therapy. However, most researchers use rats and mice as experimental models for obtaining, studying and therapeutically applying neurospheres [56,57,58,59,60]. Therefore, throughout this discussion we will focus on experimental modeling.

Table 1 Update of neural disorders using neurospheres as an in vitro model
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Among these studies, we can highlight the promising results found by Kuvacheva and collaborators (2015) who carried out a prominent study for Alzheimer's disease (AD). The study was carried out in wistar rats with the aim of evaluating the β-amyloid neurotoxicity and maintaining the development of neural progenitors. To this end, the authors evaluated the in vitro development of brain progenitor cells isolated from healthy Wistar rats and Wistar rats with Alzheimer's disease. The experimental model for AD was modeled through steroid-guided injection of β-amyloid in the CA1 field of the hippocampus. In animals with experimental AD, a slow growth of the neurosphere was observed compared to healthy animals. Subsequently, the neurosphere stabilization and expansion phase was practically absent in cells isolated from the brain of animals with AD, with the cell index gradually decreasing. These findings reveal a more intense proliferation and greater potential for neurospheres repair in healthy animals compared to the experimental model of AD [58].

In 2004, Wennersten and co-authors showed that neurospheres were strong candidates for therapeutic transplantation in neurological diseases that result in cell damage. The study evaluated the rate of proliferation, migration, and differentiation of human neural stem/progenitor cells after transplantation into a rat model of traumatic brain injury. The NSCs were obtained from 10-week-old human forebrain and, soon after a parietal cortical contusion injury was performed, the animals received the progenitor cell transplant. After a few weeks, the researchers realized that human cells were present in regions such as the perilesional zone, hippocampus and callosum body, showing that NSCs proliferate and survive after transplantation, with good migration rate and differentiation in the injured brain [60].

In neurological diseases where there is a disorder in CNS cell activity that generates recurrent symptoms, as in the case of epilepsy, the use of NSCs has been widely studied. In a model of epilepsy, Romariz et al. performed an assessment between the anticonvulsant potential and neurodifferentiation of neurospheres derived from medial ganglionic eminence (MGE) with newly isolated cells being transplanted in the hippocampus of epileptic rats. According to the authors, because they differentiate into glial cells, neurospheres derived from MGE showed anticonvulsant effects, reducing seizures in the TLE model, providing results that demonstrate that the neurosphere test becomes a cell therapy of great benefit in the treatment of epilepsy [61].

Following the same line of research, Gois da Silva et al. [62] evaluated the role of neural stem cells and their development in the brain tissue of adult epileptic Wistar rats. In epileptic rats treated with neurospheres, elevated levels of thiobarbituric acid and nitrite and a reduction in glutathione, superoxide dismutase and catalase levels were observed when compared to untreated groups. It was also possible to observe a homogeneous distribution of neurospheres throughout the brain tissue, with viable cells and in neurodifferentiation in the pilocarpine group, acting on necrosis sites, demonstrating that neurosphere therapy becomes a protective tool due to its antioxidant effect, decreasing the damage caused by seizures, helping to repair the degenerative areas [62].

NSA is a technique that can also be applied in the study of demyelinating diseases. One example was the study conducted to evaluate the potential for myelin repair by transplanting neural precursor cells derived from tissue removed during adult human brain surgery. The cultured cells showed neuronal and astrocytic characteristics that when transplanted into the spinal cord of adult rats had extensive remyelination with a pattern similar to Schwann cells. It was observed that remyelinated axons began to conduct impulses close to normal conduction velocity, suggesting that the use of neurospheres that generate neural precursors for transplantation in demyelinated areas results in satisfactory functional myelinization [63].

One issue that generates discussion among researchers is proliferation, that is, the long-term survival of transplanted cells, especially in cases of chronic diseases. As Einstein et al. reported, exposing NSCs to certain factors causes cells to perform better in differentiating neuronal lines, surviving within the ventricles, and ensuring an extensive migration to areas of inflammation. From brain tissue from mice, they obtained floating spheres that were later transplanted into the lateral ventricles of rats. With this, they demonstrated that neurospheres were able to survive even in an environment with deficiency of growth factors, responding to stimuli for proliferation and differentiation in neuronal lineage [64].

Among the various areas from which neurospheres can be used, NSCs that come from them are particularly important in the study of neurogenesis. However, nowadays it is difficult to obtain human tissue for studies with stem cells/neural precursors and with this it is essential to discover new techniques of isolation and cell culture. For this, Chang et el described a method from human amniotic liquid, liquid that contains several cells that make up the developing fetus, to isolate and proliferate human NSCs. The neurospheres, which underwent a long period of in vitro expansion, expressed specific markers for NSCs and neurons, astrocytes and oligodendrocytes. NSCs derived from amniotic liquid were grafted into the ischemic zone of the brain of rats for the purpose of functional recovery. The study reported a rapid improvement in motor function in the first week after transplantation with NSCs, stating that cells derived from amniotic fluid could easily integrate the injured ischemic area, serving as a good model in the treatment of neurodegenerative diseases [65].

Technical limitations

Although the use of neurospheres has many advantages, we need to consider their heterogeneous character and sensitivity, mainly when studying biological processes, making it difficult to compare results from different labs. There is a possibility of deregulation of the potential for differentiation and spatial identity of stem cells grown in the NSA in the presence of high concentrations of growth factors [66]. Also, there may have progressive loss of neurogenic potential with passage and low neuron yield after transplantation of expanded cells [41]. For example, spheres from neonatal mouse subependymal zone when transplanted into the lateral ventricle of mice cannot selfrenew in vivo. Also, these neurospheres do not contribute to the in vivo long-term neurogenesis [66, 67].To overcome some limitations of the NSA, Sipahi and Zupanc developed a cellular automata (CA) model. Effects of proliferative potential, contact inhibition, cell death, and clearance of dead cells on growth rate, final size, and composition of neurospheres were examined. According to the authors results a surprising prediction derived from this model is that cell death, while resulting in a decrease in growth rate and final size of neurospheres, increases the degree of differentiation of neurosphere cells. Interestingly, this approach makes it possible to simulate similarly the characteristics of spheres grown under culture conditions, being applied in a wide range of systems [68].

Another solution for the neurospheres limitations is the use of organoid technologies modeled from fibroblasts, for example, which can then be reprogrammed at an embryonic level, the so-called human induced pluripotent stem cell (hiPSC) and then differentiated into the desired cell type. The reprogramming of differentiated cells may occur by transfer of nuclear content to oocytes or by fusion with embryonic stem cells.This approach allows great potential for disease modelling, translational adoption in drug screening and regenerative medicine, and clinical research, revolutionizing the study of human brain and CNS in vitro [69, 70].

Brain organoids are widely studied because they mimic an embryonic and adult neuronal development system. This culture shows characteristics that help to mimic the study with cortical development in vivo and in vitro. For the technique of direct reprogramming of hiPSC can be used autologous cells to induce specific types of cells such as neurons, progenitors of blood cells, hepatocytes, and cartilage, also determining the destination of germ cells. Organoids technology has contributed to studies in ischemic stroke, traumatic brain injury, cell transplantation and other brain-related [71, 72].

One of the protocols developed for organoids generation was described by Lancaster and Knoblich, allowing brain organoids to be generated from reprogrammed cells of patients with different pathologies. The development of the organoids technique allowed a specific modeling for the understanding of the spectrum of some neurological disorders [73, 74]. For neuronal differentiation from this model, NSCs form a homogeneous population after some passages, resulting in more mature neural progenitors [75] The differentiation of hiPSCs in NSCs is an efficient method that generates greater differentiation in functional neurons, also allows a greater number of passages, cell freezing and thawing and manipulation in order to cell growth as neurospheres. NSCs derived from organoids are considered equally ideal as models for cellular and molecular tests in the understanding of various CNS diseases, being appropriate for studies with late characteristics of neurological diseases [76].

Currently, experimental models have been established for a better understanding of the differentiation and cell morphogenesis processes in vitro. Comparing the neurosphere assay and organoid technology, it is important to note that the neurosphere is mainly related to the expansion of NSCs, while organoids are prioritized in studies of histogenesis, differentiation and coordinated migration of NSCs. Authors report that the process of differentiating morphogenesis of the formation of neuronal clusters needs to recapitulate the specific aspects of cerebral histogenesis found in vivo and both techniques are in line with the expectations of the researchers [71, 77].

Future perspectives

For several decades research has indicated that neural tissues grafted on the CNS can repair or replace damaged neural structures and the development of characterized and functional neural precursors can be cultivated for a long period in vitro, being a beneficial alternative source for transplants. The transplantation of neural stem cells has already proved significant in the development, function, and regenerative capacities in the CNS, effectively compensating the damaged tissue.

These findings have important implications for understanding the growth characteristics, differentiation and molecular specification of neurospheres derived from the CNS, as well as the therapeutic potential of these cells for neural repair [78]. Neurospheres can be seen not only as a single cluster of cells, but as an independent "ecosystem" full of neural progenitors that will give rise to the main cells of the CNS.

Therefore, we must continually explore the NSA, as a powerful in vitro model for a better understanding of the characteristics of neural cells, their specific markers for their selection at different stages of maturation and explore their potential use for transplants in neurodegenerative syndromes, in order to obtain a satisfactory response to the patient during the course of the disease.