Hypoxia and neural stem cells: from invertebrates to brain cancer stem cells

Oxygen is a fundamental element for all living organisms, and modifications in its concentration influence several physiological and pathological events such as embryogenesis, development and also aging. Regulation of oxygen levels is an important factor in neural stem cell biology (e.g. differentiation, growth and the capacity to generate more differentiated cells). Studies on neural stem cells in culture have deepened our knowledge of their survival, proliferation and differentiation pathways. However, traditional cell culture for neural stem cells is performed employing environmental oxygen levels of 20%, while the effective oxygen concentration in the developing and adult brain is significantly lower; this results in an important alteration of the in vivo conditions. Several data indicate that a so called "physiologic hypoxic condition" could strongly influence the growth of neural stem cells and their differentiation mechanisms both in vivo and in vitro. The present overview deals with the different mechanisms utilized by invertebrate and vertebrate organisms to respond to hypoxic conditions. It highlights how the adaptations and responses to different oxygen concentrations have changed along the developmental route and underlines the importance of oxygen concentration in neural physiology and differentiation, with a final hint to the involvement of hypoxia in brain cancer stem cells.     

Brain tumour stem cells: implications for cancer therapy and regenerative medicine

The cancer relapse and mortality rate suggest that current therapies do not eradicate all malignant cells. Currently, it is accepted that tumorigenesis and organogenesis are similar in many respects, as for example, homeostasis is governed by a distinct sub-population of stem cells in both situations. There is increasing evidence that many types of cancer contain their own stem cells: cancer stem cells (CSC), which are characterized by their self-renewing capacity and differentiation ability. The investigation of solid tumour stem cells has gained momentum particularly in the area of brain tumours. Gliomas are the most common type of primary brain tumours. Nearly two-thirds of gliomas are highly malignant lesions with fast progression and unfortunate prognosis. Despite recent advances, two-year survival for glioblastoma (GBM) with optimal therapy is less than 30%. Even among patients with low-grade gliomas that confer a relatively good prognosis, treatment is almost never curative. Recent studies have demonstrated the existence of a small fraction of glioma cells endowed with features of primitive neural progenitor cells and a tumour-initiating function. In general, this fraction is characterized for forming neurospheres, being endowed with drug resistance properties and often, we can isolate some of them using sorting methods with specific antibodies. The molecular characterization of these stem populations will be critical to developing an effective therapy for these tumours with very dismal prognosis. To achieve this aim, the development of a mouse model which recapitulates the nature of these tumours is essential. This review will focus on glioma stem cell knowledge and discuss future implications in brain cancer therapy and regenerative medicine.     

Immunocytochemistry: human neural stem cells

Immunocytochemistry is a very powerful and fairly straightforward method for determining the presence, subcellular localization, and relative abundance of an antigen of interest, most commonly a protein, in cultured cells. This protocol presents an easy-to-follow series of steps that will enable researchers to conserve primary and secondary antibodies while getting high quality, reproducible qualitative and quantitative data out of their staining. There are two aspects of this protocol that help to conserve the volume of antibody necessary for staining. For one, the cells are grown on small, circular coverslips that are placed in wells of a tissue culture plate. After fixation, the cells on coverslips can be removed from the wells of the plate. For antibody staining, the coverslip with cells is inverted onto a small drop of antibody solution on parafilm and is covered with a second piece of parafilm to prevent drying. Using this method, only approximately 25 microl of antibody solution is needed for each coverslip (or sample) to be stained. This protocol describes immunostaining of human neural stem/precursor cells (hNSPCs), but can be used for many other cell types.     

Brain micro-ecologies: neural stem cell niches in the adult mammalian brain

Neurogenesis persists in two germinal regions in the adult mammalian brain, the subventricular zone of the lateral ventricles and the subgranular zone in the hippocampal formation. Within these two neurogenic niches, specialized astrocytes are neural stem cells, capable of self-renewing and generating neurons and glia. Cues within the niche, from cell-cell interactions to diffusible factors, are spatially and temporally coordinated to regulate proliferation and neurogenesis, ultimately affecting stem cell fate choices. Here, we review the components of adult neural stem cell niches and how they act to regulate neurogenesis in these regions.     

Brain as a paradigm of organ growth: Hedgehog-Gli signaling in neural stem cells and brain tumors

The Hedgehog-Gli (Hh-Gli) signaling pathway is essential for numerous events during the development of many animal cell types and organs. In particular, it controls neural cell precursor proliferation in dorsal brain structures and regulates the number of neural stem cells in distinct embryonic, perinatal, and adult niches, such as the developing neocortex, the subventricular zone of the lateral ventricle of the forebrain, and the hippocampus. We have proposed that Hh-Gli signaling regulates dorsal brain growth during ontogeny and that its differential regulation underlays evolutionary change in the morphology (size and shape) of dorsal brain structures. It is also critically involved in sporadic brain tumorigenesis--as well as several other human cancer--suggesting that tumors derive from stem cells or progenitors maintaining an inappropriate active Hh-Gli pathway. Importantly, we and others have demonstrated that human sporadic tumors from the brain and other organs require sustained HH-GLI signaling for sustained growth and survival. Modulating HH-GLI signaling thus represents a novel rational avenue to treat, on one hand, brain degeneration and injury by inducing controlled HH-GLI-mediated regeneration and growth, and on the other hand, to combat cancer by blocking its abnormal activity in tumor cells.     

Large-scale generation of human iPSC-derived neural stem cells/early neural progenitor cells and their neuronal differentiation

Induced pluripotent stem cell (iPSC)-based technologies offer an unprecedented opportunity to perform high-throughput screening of novel drugs for neurological and neurodegenerative diseases. Such screenings require a robust and scalable method for generating large numbers of mature, differentiated neuronal cells. Currently available methods based on differentiation of embryoid bodies (EBs) or directed differentiation of adherent culture systems are either expensive or are not scalable. We developed a protocol for large-scale generation of neuronal stem cells (NSCs)/early neural progenitor cells (eNPCs) and their differentiation into neurons. Our scalable protocol allows robust and cost-effective generation of NSCs/eNPCs from iPSCs. Following culture in neurobasal medium supplemented with B27 and BDNF, NSCs/eNPCs differentiate predominantly into vesicular glutamate transporter 1 (VGLUT1) positive neurons. Targeted mass spectrometry analysis demonstrates that iPSC-derived neurons express ligand-gated channels and other synaptic proteins and whole-cell patch-clamp experiments indicate that these channels are functional. The robust and cost-effective differentiation protocol described here for large-scale generation of NSCs/eNPCs and their differentiation into neurons paves the way for automated high-throughput screening of drugs for neurological and neurodegenerative diseases.     

Counting human neural stem cells

Knowledge of the exact number of viable cells in a given volume of a cell suspension is required for many routine tissue culture manipulations, such as plating cells for immunocytochemistry or for cell transfections. This protocol describes a straightforward and fast method for differentiating between live and dead cells and quantifying the cell concentration and total cell number using a hemacytometer. This procedure first requires detaching cells from a growth surface and resuspending them in media. Next, the cells are diluted in a solution of Trypan blue (ideally to a concentration that will give 20-50 cells per quadrant) and placed in the hemacytometer. Finally, averaging the counts of viable cells in several randomly selected quadrants, dividing the average by the volume of one 1 mm(2) quadrant (0.1 microl) and multiplying by the dilution factor gives the number of cells per l. Multiplying this cell concentration by the total volume in microl gives the total cell number. This protocol describes counting human neural stem/precursor cells (hNSPCs), but can also be used for many other cell types.     

Passaging human neural stem cells

The ability to manipulate human neural stem/precursor cells (hNSPCs) in vitro provides a means to investigate their utility as cell transplants for therapeutic purposes as well as to explore many fundamental processes of human neural development and pathology. This protocol presents a simple method of culturing and passaging hNSPCs in hopes of standardizing this technique and increasing reproducibility of human stem cell research. The hNSPCs we use were isolated from cadaveric postnatal brain cortices by the National Human Neural Stem Cell Resource and grown as adherent cultures on flasks coated with fibronectin (Palmer et al., 2001; Schwartz et al., 2003). We culture our hNSPCs in a DMEM:F12 serum-free media supplemented with EGF, FGF, and PDGF and passage them 1:2 approximately every seven days. Using these conditions, the majority of the cells in the culture maintain a bipolar morphology and express markers of undifferentiated neural stem cells (such as nestin and sox2).     

Brain injury expands the numbers of neural stem cells and progenitors in the SVZ by enhancing their responsiveness to EGF

There is an increase in the numbers of neural precursors in the SVZ (subventricular zone) after moderate ischaemic injuries, but the extent of stem cell expansion and the resultant cell regeneration is modest. Therefore our studies have focused on understanding the signals that regulate these processes towards achieving a more robust amplification of the stem/progenitor cell pool. The goal of the present study was to evaluate the role of the EGFR [EGF (epidermal growth factor) receptor] in the regenerative response of the neonatal SVZ to hypoxic/ischaemic injury. We show that injury recruits quiescent cells in the SVZ to proliferate, that they divide more rapidly and that there is increased EGFR expression on both putative stem cells and progenitors. With the amplification of the precursors in the SVZ after injury there is enhanced sensitivity to EGF, but not to FGF (fibroblast growth factor)-2. EGF-dependent SVZ precursor expansion, as measured using the neurosphere assay, is lost when the EGFR is pharmacologically inhibited, and forced expression of a constitutively active EGFR is sufficient to recapitulate the exaggerated proliferation of the neural stem/progenitors that is induced by hypoxic/ischaemic brain injury. Cumulatively, our results reveal that increased EGFR signalling precedes that increase in the abundance of the putative neural stem cells and our studies implicate the EGFR as a key regulator of the expansion of SVZ precursors in response to brain injury. Thus modulating EGFR signalling represents a potential target for therapies to enhance brain repair from endogenous neural precursors following hypoxic/ischaemic and other brain injuries.     

Deadly teamwork: neural cancer stem cells and the tumor microenvironment

Neural cancers display cellular hierarchies with self-renewing tumorigenic cancer stem cells (CSCs) at the apex. Instructive cues to maintain CSCs are generated by both intrinsic networks and the niche microenvironment. The CSC-microenvironment relationship is complex, as CSCs can modify their environment and extrinsic forces induce plasticity in the cellular hierarchy.     

Adult neural stem cells bridge their niche

Major developments in the neural stem cell (NSC) field in recent years provide new insights into the nature of the NSC niche. In this perspective, we integrate recent anatomical data on the organization of the two main neurogenic niches in the adult brain, the ventricular-subventricular zone (V-SVZ) and the subgranular zone (SGZ), with signaling pathways that control the behavior of NSCs. NSCs in the adult brain stretch into physiologically distinct compartments of their niche. We propose how adult NSCs' morphology may allow these cells to integrate multiple signaling pathways arising from unique locations of their niche.     

Differentiation profile of brain tumor stem cells： a comparative study with neural stem cells

Understanding of the differentiation profile of brain tumor stem cells （BTSCs）, the key ones among tumor cell population, through comparison with neural stem cells （NSCs） would lend insight into the origin of glioma and ultimately yield new approaches to fight this intractable disease. Here, we cultured and purified BTSCs from surgical glioma specimens and NSCs from human fetal brain tissue, and further analyzed their cellular biological behaviors, especially their differentiation property. As expected, NSCs differentiated into mature neural phenotypes. In the same differentiation condition, however, BTSCs exhibited distinguished differences. Morphologically, cells grew flattened and attached for the first week, but gradually aggregated and reformed floating tumor sphere thereafter. During the corresponding period, the expression rate of undifferentiated cell marker CD 133 and nestin in BTSCs kept decreasing, but 1 week later, they regained ascending tendency. Interestingly, the differentiated cell markers GFAP and β-tubulinlII showed an expression change inverse to that of undifferentiated cell markers. Taken together, BTSCs were revealed to possess a capacity to resist differentiation, which actually represents the malignant behaviors of glioma.     

On the proportion of cancer stem cells in a tumour

It is now generally accepted that cancers contain a sub-population, the cancer stem cells (CSCs), which initiate and drive a tumour’s growth. At least until recently it has been widely assumed that only a small proportion of the cells in a tumour are CSCs. Here we use a mathematical model, supported by experimental evidence, to show that such an assumption is unwarranted. We show that CSCs may comprise any possible proportion of the tumour, and that the higher the proportion the more aggressive the tumour is likely to be.     

Long-term survival of human neural stem cells in the ischemic rat brain upon transient immunosuppression

Understanding the physiology of human neural stem cells (hNSCs) in the context of cell therapy for neurodegenerative disorders is of paramount importance, yet large-scale studies are hampered by the slow-expansion rate of these cells. To overcome this issue, we previously established immortal, non-transformed, telencephalic-diencephalic hNSCs (IhNSCs) from the fetal brain. Here, we investigated the fate of these IhNSC's immediate progeny (i.e. neural progenitors; IhNSC-Ps) upon unilateral implantation into the corpus callosum or the hippocampal fissure of adult rat brain, 3 days after global ischemic injury. One month after grafting, approximately one fifth of the IhNSC-Ps had survived and migrated through the corpus callosum, into the cortex or throughout the dentate gyrus of the hippocampus. By the fourth month, they had reached the ipsilateral subventricular zone, CA1-3 hippocampal layers and the controlateral hemisphere. Notably, these results could be accomplished using transient immunosuppression, i.e administering cyclosporine for 15 days following the ischemic event. Furthermore, a concomitant reduction of reactive microglia (Iba1+ cells) and of glial, GFAP+ cells was also observed in the ipsilateral hemisphere as compared to the controlateral one. IhNSC-Ps were not tumorigenic and, upon in vivo engraftment, underwent differentiation into GFAP+ astrocytes, and β-tubulinIII+ or MAP2+ neurons, which displayed GABAergic and GLUTAmatergic markers. Electron microscopy analysis pointed to the formation of mature synaptic contacts between host and donor-derived neurons, showing the full maturation of the IhNSC-P-derived neurons and their likely functional integration into the host tissue. Thus, IhNSC-Ps possess long-term survival and engraftment capacity upon transplantation into the globally injured ischemic brain, into which they can integrate and mature into neurons, even under mild, transient immunosuppressive conditions. Most notably, transplanted IhNSC-P can significantly dampen the inflammatory response in the lesioned host brain. This work further supports hNSCs as a reliable and safe source of cells for transplantation therapy in neurodegenerative disorders.     

内源性神经干细胞与脑老化的治疗

近十几年研究发现成年人脑神经元可以再生,使人们重新认识老年脑神经细胞的可塑性,它为脑损伤的修复带来新的希望。最新研究表明,神经再生可被调控,是一种新的修复机制。这使得利用内源性神经干细胞治疗老龄相关的神经退行性疾病成为可能。     

Long-term cultured human neural stem cells undergo spontaneous transformation to tumor-initiating cells

In this report, we describe the spontaneous malignant transformation of long-term cultured human fetal striatum neural stem cells (hsNSCs, passage 17). After subcutaneous transplantation of long-term cultured hsNSCs into immunodeficient nude mice, 2 out of 15 mice formed xenografts which expressed neuroendocrine tumor markers CgA and NSE. T1 cells, a cell line that we derived from one of the two subcutaneous xenografts, have undergone continuous expansion in vitro. These T1 cells showed stem cell-like features and expressed neural stem cell markers nestin and CD133. The T1 cells were involved in abnormal karyotype, genomic instability and fast proliferation. Importantly, after long-term in vitro culture, the T1 cells did not result in subcutaneous xenografts, but induced intracranial tumor formation, indicating that they adjusted themselves to the intracranial microenvironment. We further found that the T1 cells exhibited an overexpressed level of EGFR, and the CD133 positive T1 cells showed a truncation mutation in the exons 2-7 of the EGFR (EGFRvIII) gene. These results suggest that continuous expansion of neural stem cells in culture may lead to malignant spontaneous transformation. This phenomenon may be functionally related to EGFR by EGFRvIII gene mutation.     

Dopaminergic differentiation of human neural stem cells mediated by co-cultured rat striatal brain slices

Properly committed neural stem cells constitute a promising source of cells for transplantation in Parkinson's disease, but a protocol for controlled dopaminergic differentiation is not yet available. To establish a setting for identification of secreted neural compounds promoting dopaminergic differentiation, we co-cultured cells from a human neural forebrain-derived stem cell line (hNS1) with rat striatal brain slices. In brief, coronal slices of neonatal rat striatum were cultured on semiporous membrane inserts placed in six-well trays overlying monolayers of hNS1 cells. After 12 days of co-culture, large numbers of tyrosine hydroxylase (TH)-immunoreactive, catecholaminergic cells could be found underneath individual striatal slices. Cell counting revealed that up to 25.3% (average 16.1%) of the total number of cells in these areas were TH-positive, contrasting a few TH-positive cells (<1%) in non-induced areas. The presence of dopamine in the conditioned culture medium was confirmed by HPLC analysis. Interestingly, not all striatal slice cultures induced TH-expression in underlying hNS1 cells. Common to TH-inductive cultures was, however, the presence of degenerating, necrotic areas, suggesting that factors released during striatal degeneration were responsible for the dopaminergic induction of the hNS1 cells. Ongoing experiments aim to identify such factors by comparing protein profiles of media conditioned by degenerating (necrotic) versus healthy striatal slice cultures.