Human Fetal Neural Stem Cells Research Articles

SC-05 * DOPAMINE RECEPTOR ANTAGONISTS ARE SELECTIVE INHIBITORS OF GLIOBLASTOMA STEM CELLS THROUGH IMPAIRMENT OF AUTOPHAGY

Glioblastoma is an incurable cancer. The tumorigenic potential of glioblastoma resides within cells with a stem cell phenotype called glioblastoma stem cells. In order to identify novel glioblastoma drugs, we interrogated a library of 680 neuromodulatory compounds for their activity on glioblastoma derived neural stem cells (GNS), normal human fetal neural stem (NS) cells and normal fibroblasts to identify selective inhibitors. Compounds modulating dopaminergics, serotonergics and cholinergics pathways are enriched in the hits. In particular, two dopamine receptor antagonists demonstrated great selectivity for GNS cells and were synergistic with the chemotherapeutic drug temozolomide. These compounds reduced the clonogenic potential of primary glioblastoma cells and the growth of glioblastoma xenografts. Primary glioblastoma tumor and GNS cells expressed dopamine receptor. Mechanistic studies by pharmalogical inhibition and genetic knockdown of dopamine receptor suppressed glioblastoma stem cells growth and the ERK1/2 pathway. Furthermore, genome wide expression array showed downregulation of genes involved in cell cycle progression and upregulation of genes involved in autophagy. Further investigation revealed accumulations of autophagic vacuole with increased p62 suggesting a block in autophagy leading to cell death. This study identified new candidates for glioblastoma therapy and suggests neurochemical modifications should be focus for further drug development.

M18 Spatio-temporal Characterisation Of Human Fetal Neural Stem Cells For Cell Replacement Therapies

Human fetal neural stem cells (hfNSC) hold great potential for cell therapy strategies but further studies are still needed for a proper understanding of these cells. In the present work we characterised hfNSC isolated from cortex, whole ganglionic eminence (WGE), cerebellum and forebrain at several developmental stages (6–12 weeks post conception (wpc)). We showed that tissue obtained from younger fetuses survived significantly better than older ones after dissociation, due to the presence of a higher number Nestin positive neural precursors. These results suggested the presence of more differentiated cells in samples obtained from older fetuses. In agreement, an increased expression of mature neuronal markers was observed in samples from older fetal brains. Interestingly, the proliferation ratios were not different between developmental stages. A common feature was observed for all the cultures studied independently of the brain area and the developmental stage; they went through 3–4 initial critical passages characterised by high percentages of cell death and substantial variability in the proliferation ratio. After this critical period, they stabilised, reduced cell death and maintained a constant expansion potential. Forebrain hfNSC were the most readily expandable and cerebellar cells the ones that proliferated least. Finally, we analysed the regional identity and differentiation potential of cortical and WGE derived hfNSC along passages. The expression of mature region specific markers after 3 and 6 passages was down-regulated. Interestingly, cortical cultures acquire a ventral phenotype along passages. Nevertheless, they both keep the multipotency after 6 passages and the potential to differentiate to mature region specific neurons. In conclusion, here we show that hfNSCs can be expanded in vitro and may be suitable for cell therapy in HD.

MODELLING HIGH MYC MEDULLOBLASTOMA AND OTHER BRAIN TUMORS USING HUMAN NEURAL STEM CELLS

BACKGROUND: We used human fetal neural stem cells dissected from the cerebral cortex, thalamus, cerebellum and brainstem, then propagated as neurospheres, to model a range of brain tumors. Various combinations of oncogenic elements characteristic of medulloblastoma and glioblastoma were introduced into stem cells derived from these different sites. Transformation of cells was characterized in vitro and in vivo, and MYC driven medulloblastoma were used for preclinical testing of glutaminase inhibitors. METHODS: We transduced cerebellar derived human neural stem cells with lentivirus coding for MYC, dominant negative R248W p53, constitutively active AKT, and hTERT in several combinations. Growth of transformed cells and their response to therapy was analyzed in vitro and in vivo using standard techniques. RESULTS: MYC alone was sufficient to transform cerebellar progenitor cells into embryonal tumors with a predominantly neuronal phenotype, but with a long latency period and decreased penetrance suggesting secondary genetic changes were required. The R248Wp53/MYC/AKT/hTERT combination transformed cerebellar stem cells into more aggressive orthotopic xenografts with large cell/anaplastic histology and spinal metastases. Microarray expression analysis suggested that these tumors represent a model of high MYC group 3 medulloblastoma. MYC expression positively correlated with increased expression of glutaminase, and the glutaminase inhibitor Acivicin decreased cell proliferation more strongly in MYC-containing neurospheres compared to those without MYC. Acivicin treatment of MYC expressing cells also led to a much greater increase in apoptosis compared to MYC-negative cells. Introduction of other packages of oncogenic elements into cortical and thalamic neurospheres resulted in PNET-like tumors, including variants resembling medulloepithelioma, as well as glioblastoma. CONCLUSIONS: Human cerebellar stem cells can be transformed by c-MYC and cooperating oncogenes, resulting in a models that recapitulates group 3 medulloblastoma histologically and clinically. These cells and classical high-MYC medulloblastoma cell lines are sensitive to glutamine anti-metabolites, while non-MYC driven medulloblastoma and SV40 immortalized human cerebellar stem cells are resistant. Our group 3 human neural stem cell medulloblastoma model data suggest that glutamine metabolism may be a therapeutic target in MYC-driven medulloblastoma, and that glutamine anti-metabolites may be useful clinical agents. Neurospheres from other CNS sites can be transformed with other oncogenes into models of PNET and glioblastoma. SECONDARY CATEGORY: Tumor Biology.

Human neural stem cells survive long term in the midbrain of dopamine-depleted monkeys after GDNF overexpression and project neurites toward an appropriate target.

Transplanted multipotent human fetal neural stem cells (hfNSCs) significantly improved the function of parkinsonian monkeys in a prior study primarily by neuroprotection, with only 3%-5% of cells expressing a dopamine (DA) phenotype. In this paper, we sought to determine whether further manipulation of the neural microenvironment by overexpression of a developmentally critical molecule, glial cell-derived neurotrophic factor (GDNF), in the host striatum could enhance DA differentiation of hfNSCs injected into the substantia nigra and elicit growth of their axons to the GDNF-expressing target. hfNSCs were transplanted into the midbrain of 10 green monkeys exposed to 1-methyl-4-phenyl-1,2,3,6-tetrahydro-pyridine. GDNF was delivered concomitantly to the striatum via an adeno-associated virus serotype 5 vector, and the fate of grafted cells was assessed after 11 months. Donor cells remained predominantly within the midbrain at the injection site and sprouted numerous neurofilament-immunoreactive fibers that appeared to course rostrally toward the striatum in parallel with tyrosine hydroxylase-immunoreactive fibers from the host substantia nigra but did not mature into DA neurons. This work suggests that hfNSCs can generate neurons that project long fibers in the adult primate brain. However, in the absence of region-specific signals and despite GDNF overexpression, hfNSCs did not differentiate into mature DA neurons in large numbers. It is encouraging, however, that the adult primate brain appeared to retain axonal guidance cues. We believe that transplantation of stem cells, specifically instructed ex vivo to yield DA neurons, could lead to reconstruction of some portion of the nigrostriatal pathway and prove beneficial for the parkinsonian condition.

Human Fetal Striatum-Derived Neural Stem (NS) Cells Differentiate to Mature Neurons In Vitro and In Vivo

Clonogenic neural stem (NS) cell lines grown in adherent cultures have previously been established from embryonic stem cells and fetal and adult CNS in rodents and from human fetal brain and spinal cord. Here we describe the isolation of a new cell line from human fetal striatum (hNS cells). These cells showed properties of NS cells in vitro such as monolayer growth, high proliferation rate and expression of radial glia markers. The hNS cells expressed an early neuronal marker while being in the proliferative state. Under appropriate conditions, the hNS cells were efficiently differentiated to neurons, and after 4 weeks about 50% of the cells were βIII tubulin positive. They also expressed the mature neuronal marker NeuN and markers of neuronal subtypes, GABA, calbindin, and DARPP32. After intrastriatal implantation into newborn rats, the hNS cells survived and many of them migrated outside the transplant core into the surrounding tissue. A high percentage of cells in the grafts expressed the neuroblast marker DCX, indicating their neurogenic potential, and some of the cells differentiated to NeuN+ mature neurons. The human fetal striatum-derived NS cell line described here should be a useful tool for studies on cell replacement strategies in models of the striatal neuronal loss occurring in Huntington's disease and stroke.

Neural stem cell-conditioned medium protects neurons and promotes propriospinal neurons relay neural circuit reconnection after spinal cord injury.

Human fetal neural stem cells (hNSCs) are used to treat a variety of neurological disorders involving spinal cord injury (SCI). Although their mechanism of action has been attributed to cell substitution, we examined the possibility that NSCs may have neuroprotective activities. The present article studied the action of hNSCs on protecting neurons and promoting corticospinal tract (CST) axon regeneration after SCI. hNSCs were isolated from the cortical tissue of spontaneously aborted human fetuses. The cells were removed from the NSC culture medium to acquire NSCM, thus excluding the effect of cell substitution. Continuous administration of the NSCM after the SCI resulted in extensive growth of the CST in the cervical region and more than tripled the formation of synaptic contacts between CST collaterals and propriospinal interneurons that project from the cervical level of the spinal cord to the lumbar level. NSCM reduced the number of caspase 3-positive apoptotic profiles at 7 days and protected against loss of the neurons 6 weeks after injury. NSCM promoted locomotor recovery with a five-point improvement on the BBB scale in adult rats. Thus, hNSCs help to set up a contour neural circuit via secretory factors, which may be the mechanism for their action in SCI rats. This manuscript is published as part of the International Association of Neurorestoratology (IANR) special issue of Cell Transplantation.

Derivation of Neural Stem Cells from Human Adult Peripheral CD34+ Cells for an Autologous Model of Neuroinflammation

Proinflammatory factors from activated T cells inhibit neurogenesis in adult animal brain and cultured human fetal neural stem cells (NSC). However, the role of inhibition of neurogenesis in human neuroinflammatory diseases is still uncertain because of the difficulty in obtaining adult NSC from patients. Recent developments in cell reprogramming suggest that NSC may be derived directly from adult fibroblasts. We generated NSC from adult human peripheral CD34+ cells by transfecting the cells with Sendai virus constructs containing Sox2, Oct3/4, c-Myc and Klf4. The derived NSC could be differentiated to glial cells and action potential firing neurons. Co-culturing NSC with activated autologous T cells or treatment with recombinant granzyme B caused inhibition of neurogenesis as indicated by decreased NSC proliferation and neuronal differentiation. Thus, we have established a unique autologous in vitro model to study the pathophysiology of neuroinflammatory diseases that has potential for usage in personalized medicine.

Human neural stem cell transplantation in spinal cord injury models: how far from clinical application?

Injuries of the spinal cord trigger local healing but hardly restore normal function. Spinal cord injury (SCI) has been deeply studied to develop strategies for functional recovery. The study by van Gorp and colleagues uses a rat model of acute SCI to characterize the effects of intraspinal grafting of human fetal spinal cord-derived neural stem cells (HSSCs). Among the range of results obtained, several positive outcomes related to a GABA-mediated inhibition were achieved. Their interesting article is a clear step forward in elucidating the neurobiology of SCI. Some issues to consider are related to the efficacy of the cell therapy in chronic spinal lesions and to pain responses, since the latter is a common complaint of people with SCI. Furthermore, the GABA-mediated hypothesis of recovery of function upon HSSC transplant needs to be fully tested by interfering with GABA receptors and analyzing neurobiological mechanisms that may invert the inhibitory role of GABA. The future challenge for SCI work will be the translation of knowledge acquired in basic research into clinically applicable strategies. The study by van Gorp and colleagues is an important contribution to that future.

Amelioration of motor/sensory dysfunction and spasticity in a rat model of acute lumbar spinal cord injury by human neural stem cell transplantation

IntroductionIntraspinal grafting of human neural stem cells represents a promising approach to promote recovery of function after spinal trauma. Such a treatment may serve to: I) provide trophic support to improve survival of host neurons; II) improve the structural integrity of the spinal parenchyma by reducing syringomyelia and scarring in trauma-injured regions; and III) provide neuronal populations to potentially form relays with host axons, segmental interneurons, and/or α-motoneurons. Here we characterized the effect of intraspinal grafting of clinical grade human fetal spinal cord-derived neural stem cells (HSSC) on the recovery of neurological function in a rat model of acute lumbar (L3) compression injury.MethodsThree-month-old female Sprague–Dawley rats received L3 spinal compression injury. Three days post-injury, animals were randomized and received intraspinal injections of either HSSC, media-only, or no injections. All animals were immunosuppressed with tacrolimus, mycophenolate mofetil, and methylprednisolone acetate from the day of cell grafting and survived for eight weeks. Motor and sensory dysfunction were periodically assessed using open field locomotion scoring, thermal/tactile pain/escape thresholds and myogenic motor evoked potentials. The presence of spasticity was measured by gastrocnemius muscle resistance and electromyography response during computer-controlled ankle rotation. At the end-point, gait (CatWalk), ladder climbing, and single frame analyses were also assessed. Syrinx size, spinal cord dimensions, and extent of scarring were measured by magnetic resonance imaging. Differentiation and integration of grafted cells in the host tissue were validated with immunofluorescence staining using human-specific antibodies.ResultsIntraspinal grafting of HSSC led to a progressive and significant improvement in lower extremity paw placement, amelioration of spasticity, and normalization in thermal and tactile pain/escape thresholds at eight weeks post-grafting. No significant differences were detected in other CatWalk parameters, motor evoked potentials, open field locomotor (Basso, Beattie, and Bresnahan locomotion score (BBB)) score or ladder climbing test. Magnetic resonance imaging volume reconstruction and immunofluorescence analysis of grafted cell survival showed near complete injury-cavity-filling by grafted cells and development of putative GABA-ergic synapses between grafted and host neurons.ConclusionsPeri-acute intraspinal grafting of HSSC can represent an effective therapy which ameliorates motor and sensory deficits after traumatic spinal cord injury.

Stimulation of cholinogenesis in human fetal nerve cell cultures

The aim of the study was to obtain a neural cell population enriched in culture with cholinergic neurons and their determined precursors. It has been established that the joint application of retinoic acid and acetylcholine appears to be the most efficient combination of neuroinductors, stimulating the differentiation of cholinergic neurons from neural stem cells. During the entire period of culturing, the amount of ChAT+-positive cells increased reliably to 21.1 ± 6.2% from 5.3 ± 2.9, whereas the ir concentration in the control samples was 9.1 ± 4.8% of the total cell count. The cell population enriched with cholinergic neurons and their determined precursors correlated well with the increased acetylcholinesterase activity. Thus, the addition of retinoic acid and acetylcholine stimulate both neurogenesis and cholinogenesis in human fetal neural stem cell cultures.

Treating spinal cord injury in rats with a combination of human fetal neural stem cells and hydrogels modified with serotonin.

Currently, there is no effective strategy for the treatment of spinal cord injury (SCI). A combination of biomaterials and stem cell therapy seems to be a promising approach to increase regenerative potential after SCI. We evaluated the use of a cellpolymer construct based on a combination of the conditionally immortalized spinal progenitor cell line SPC-01_GFP3, derived from human fetal spinal cord tissue, with a serotonin-modified poly(2-hydroxyethyl methacrylate) hydrogel (pHEMA-5HT). We compared the effect of treatment with a pHEMA-5HT hydrogel seeded with SPC-01_GFP3 cells, treatment with a pHEMA-5HT only and no treatment on functional outcome and tissue reconstruction in hemisected rats. Prior to transplantation the cell-polymer construct displayed a high potential to support the growth, proliferation and differentiation of SPC-01 cells in vitro. One month after surgery, combined hydrogel-cell treatment reduced astrogliosis and tissue atrophy and increased axonal and blood vessel ingrowth into the implant; however, two months later only the ingrowth of blood vessels remained increased. SPC-01_GFP3 cells survived well in vivo and expressed advanced markers of neuronal differentiation. However, a majority of the transplanted cells migrated out of the lesion and only rarely remained in the hydrogel. No differences among the groups in motor or sensory recovery were observed. Despite the support of the hydrogel as a cell carrier in vitro, and good results in vivo one month postsurgery, there was only a small effect on long term recovery, mainly due to the limited ability of the hydrogels to support the in vivo growth and differentiation of cells within the implant. Further modifications will be necessary to achieve stable long term improvement in functional outcome.

Combined Transplantation of Human Neuronal and Mesenchymal Stem Cells following Spinal Cord Injury

Transplantation of human fetal neural stem cells (hNSCs) previously demonstrated significant functional recovery after spinal cord contusion in rats. Other studies indicated that human mesenchymal stem cells (hMSCs) can home to areas of damage and cross the blood–brain barrier. The purpose of this article is to determine if combined administration of mesenchymal stem cells and neuronal stem cells improves functional outcomes in rats. The study design was a randomized controlled animal trial. Female adult Long-Evans hooded rats underwent laminectomy at T10 level. Moderate spinal cord contusion at T10 level was induced by the MASCIS Impactor. Four groups were identified. The MSC + NSC group received hMSCs intravenously (IV) immediately after spinal cord injury (acute) and returned 1 week later (subacute) for injection of hNSC directly at site of injury. The MSC-only group received hMSC IV acutely and cell media subacutely. The NSC-only group received cell media IV acutely and hNSC subacutely. The control group received cell media IV acutely and subacutely. Subjects were assessed for 6 weeks using Basso, Beattie, Bresnahan Locomotor Rating Score. Twenty-four subjects were utilized, six subjects in each group. Statistically significant functional improvement was seen in the MSC + NSC group and the NSC-only group versus controls (p = 0.027, 0.042, respectively). The MSC-only group did not demonstrate a significant improvement over control (p = 0.145). Comparing the MSC + NSC group and the NSC-only group, there was no significant difference (p = 0.357). Subacute transplantation of hNSCs into contused spinal cord of rats led to significant functional recovery when injected either with or without acute IV administration of hMSCs. Neither hMSCs nor addition of hMSC to hNSC resulted in significant improvement.

Functional assessment of the acute local and distal transplantation of human neural stem cells after spinal cord injury

Spinal cord injury can lead to severe functional impairments secondary to axonal damage, neuronal loss, and demyelination. The injured spinal cord has limited regrowth of damaged axons. Treatment remains controversial, given inconsistent functional improvement. Previous studies demonstrated functional recovery of rats with spinal cord contusion after transplantation of rat fetal neural stem cells. We hypothesized that acute transplantation of human fetal neural stem cells (hNSCs) both locally at the injury site as well as distally via intrathecal injection would lead to improved functional recovery compared with controls. Twenty-four adult female Long-Evans hooded rats were randomized into four groups with six animals in each group: two experimental and two control. Functional assessment was measured after injury and then weekly for 6 weeks using the Basso, Beattie, and Bresnahan Locomotor Rating Score. Data were analyzed using two-sample t test and linear mixed-effects model analysis. Posterior exposure and laminectomy at T10 level was used. Moderate spinal cord contusion was induced by the Multicenter Animal Spinal Cord Injury Study Impactor with 10-g weight dropped from a height of 25 mm. Experimental subjects received either a subdural injection of hNSCs locally at the injury site or intrathecal injection of hNSCs through a separate distal laminotomy. Controls received control media injection either locally or distally. Statistically significant functional improvement was observed in local or distal hNSCs subjects versus controls (p=.034 and 0.016, respectively). No significant difference was seen between local or distal hNSC subjects (p=.66). Acute local and distal transplantation of hNSCs into the contused spinal cord led to significant functional recovery in the rat model. No statistical difference was found between the two techniques.

Efficient expansion and dopaminergic differentiation of human fetal ventral midbrain neural stem cells by midbrain morphogens

Human fetal midbrain tissue grafting has provided proof-of-concept for dopamine cell replacement therapy (CRT) in Parkinson's disease (PD). However, limited tissue availability has hindered the development and widespread use of this experimental therapy. Here we present a method for generating large numbers of midbrain dopaminergic (DA) neurons based on expanding and differentiating neural stem/progenitor cells present in the human ventral midbrain (hVM) tissue. Our results show that hVM neurospheres (hVMN) with low cell numbers, unlike their rodent counterparts, expand the total number of cells 3-fold, whilst retaining their capacity to differentiate into midbrain DA neurons. Moreover, Wnt5a promoted DA differentiation of expanded cells resulting in improved morphological maturation, midbrain DA marker expression, DA release and electrophysiological properties. This method results in cell preparations that, after expansion and differentiation, can contain 6-fold more midbrain DA neurons than the starting VM preparation. Thus, our results provide evidence that by improving expansion and differentiation of progenitors present in the hVM it is possible to greatly enrich cell preparations for DA neurons. This method could substantially reduce the amount of human fetal midbrain tissue necessary for CRT in patients with PD, which could have major implications for the widespread adoption of this approach.

Human Neural Stem Cell Replacement Therapy for Amyotrophic Lateral Sclerosis by Spinal Transplantation

BackgroundMutation in the ubiquitously expressed cytoplasmic superoxide dismutase (SOD1) causes an inherited form of Amyotrophic Lateral Sclerosis (ALS). Mutant synthesis in motor neurons drives disease onset and early disease progression. Previous experimental studies have shown that spinal grafting of human fetal spinal neural stem cells (hNSCs) into the lumbar spinal cord of SOD1G93A rats leads to a moderate therapeutical effect as evidenced by local α-motoneuron sparing and extension of lifespan. The aim of the present study was to analyze the degree of therapeutical effect of hNSCs once grafted into the lumbar spinal ventral horn in presymptomatic immunosuppressed SOD1G93A rats and to assess the presence and functional integrity of the descending motor system in symptomatic SOD1G93A animals.Methods/Principal FindingsPresymptomatic SOD1G93A rats (60–65 days old) received spinal lumbar injections of hNSCs. After cell grafting, disease onset, disease progression and lifespan were analyzed. In separate symptomatic SOD1G93A rats, the presence and functional conductivity of descending motor tracts (corticospinal and rubrospinal) was analyzed by spinal surface recording electrodes after electrical stimulation of the motor cortex. Silver impregnation of lumbar spinal cord sections and descending motor axon counting in plastic spinal cord sections were used to validate morphologically the integrity of descending motor tracts. Grafting of hNSCs into the lumbar spinal cord of SOD1G93A rats protected α-motoneurons in the vicinity of grafted cells, provided transient functional improvement, but offered no protection to α-motoneuron pools distant from grafted lumbar segments. Analysis of motor-evoked potentials recorded from the thoracic spinal cord of symptomatic SOD1G93A rats showed a near complete loss of descending motor tract conduction, corresponding to a significant (50–65%) loss of large caliber descending motor axons.Conclusions/SignificanceThese data demonstrate that in order to achieve a more clinically-adequate treatment, cell-replacement/gene therapy strategies will likely require both spinal and supraspinal targets.

Longitudinal evaluation of MPIO-labeled stem cell biodistribution in glioblastoma using high resolution and contrast-enhanced MR imaging at 14.1 tesla.

To optimize the development of stem cell (SC)-based therapies for the treatment of glioblastoma (GBM), we compared the pathotropism of 2 SC sources, human mesenchymal stem cells (hMSCs) and fetal neural stem cells (fNSCs), toward 2 orthotopic GBM models, circumscribed U87vIII and highly infiltrative GBM26. High resolution and contrast-enhanced (CE) magnetic resonance imaging (MRI) were performed at 14.1 Tesla to longitudinally monitor the in vivo location of hMSCs and fNSCs labeled with the same amount of micron-size particles of iron oxide (MPIO). To assess pathotropism, SCs were injected in the contralateral hemisphere of U87vIII tumor-bearing mice. Both MPIO-labeled SC types exhibited tropism to tumors, first localizing at the tumor edges, then in the tumor masses. MPIO-labeled hMSCs and fNSCs were also injected intratumorally in mice with U87vIII or GBM26 tumors to assess their biodistribution. Both SC types distributed throughout the tumor in both GBM models. Of interest, in the U87vIII model, areas of hyposignal colocalized first with the enhancing regions (ie, regions of high vascular permeability), consistent with SC tropism to vascular endothelial growth factor. In the GBM26 model, no rim of hyposignal was observed, consistent with the infiltrative nature of this tumor. Quantitative analysis of the index of dispersion confirmed that both MPIO-labeled SC types longitudinally distribute inside the tumor masses after intratumoral injection. Histological studies confirmed the MRI results. In summary, our results indicate that hMSCs and fNSCs exhibit similar properties regarding tumor tropism and intratumoral dissemination, highlighting the potential of these 2 SC sources as adequate candidates for SC-based therapies.

CD133-Positive Cells Might Be Responsible for Efficient Proliferation of Human Meningioma Cells

Owing to lack of appropriate model systems, investigations of meningioma biology have come to a stop. In this study, we developed a comprehensive digestion method and defined a culture system. Using this method and system, primary meningioma cells in conditioned suspension medium and a hypoxic environment could be amplified in spheres and were passaged for more than ten generations. Meningioma sphere cells were positive for meningioma cell markers and negative for markers of neural cell types. Importantly, we found the cells expressed the stem cell marker, CD133, but not nestin. All of the tumor sphere cell populations showed a slower degree of cell proliferation than that of human glioma cells and fetal neural stem cells (NSCs). Further studies showed that the proliferative rate was positively correlated with CD133 expression. The higher the CD133 expression, the faster the cell proliferation. With the increase in cell generations, the cell proliferation rate gradually slowed down, and CD133 expression also decreased. Single CD133+ cells rather than CD133− cells could form spheres. Thus, the results above indicated that those cells expressing CD133 in spheres might be stem-like cells, which may be responsible for efficient amplification of human meningioma cells. Decreased expression of CD133 may lead to the failure of long-term passaging.

Abstract 347: The role of the epithelial-mesenchymal transition in the maintenance of stemness in neural and glioma stem cells

Abstract Despite advances in treatment, Glioblastoma multiforme (GBM) continues to carry a horrible prognosis with an average overall survival of only 14 months [Stupp et al, 2005]. GBMs are heterogeneous tumors that contain a subpopulation of stem-cell like cells called glioblastoma stem cells (GSCs). GSCs appear to be chemotherapy- and radiation-resistant and are thought to be responsible for GBM progression [Kang et al, 2007; Bao et al, 2006]. More effective GBM therapies must target GSCs, underscoring the importance of understanding GSC biology. Our study examines the epithelial-mesenchymal transition (EMT) and its converse, the mesenchymal-epithelial transition (MET), in the context of GSCs and neural stem cells (NSCs). We hypothesize that the zinc-finger enhancer binding transcription factor (ZEB)/miR-200 feedback loop is relevant in the context of GSCs and is modulated by TGF-β and BMP. We have found that ZEB1 and miR-200 define divergent glioma cell populations: qRT-PCR shows differential expression of ZEB1 and miR-200 in mature human astrocytes, human fetal NSC, and human GSCs (GliNS1). In GSCs, BMP antagonism (noggin) or TGF-β stimulation results in increased ZEB1 expression. Conversely, BMP4 stimulation increases miR-200b/c, while noggin or TGF-β treatment decrease miR-200b/c expression. BrdU proliferation studies show increased proliferation with TGF-β stimulation and decreased proliferation with BMP treatment in three GSC lines. TGF-β signaling also confers an invasive phenotype in GSCs while BMP4 treatment decreases invasion, suggesting a role for TGF-β and BMP parallel to that seen in EMT and MET. Western blot analysis reveals that treatment of GSCs with TGF-β induces phosphorylation of Stat3, which has been shown to be a driver of EMT [Carro MS, 2010]. Interestingly, BMP and TGF-β treatment does not change Sox2 protein expression, suggesting that the GliNS1 GSCs retain their stem cell identity. However, Bmi1 increases with BMP stimulation and decreases with TGF-β treatment, while ID1 increases with BMP treatment, suggesting that the stem-ness in GSCs is modulated by these cytokines. In conclusion, BMP and TGF-β control the ZEB1/miR-200 feedback loop in GSCs and affect cell proliferation and invasive capability. We are continuing studies to determine the relevance of TGF-β and BMP signaling to patients with GBM. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 347. doi:1538-7445.AM2012-347

Expression analysis of pluripotency-associated genes in human fetal cortical and striatal neural stem cells during differentiation

AbstractIn the field of developmental biology, there is compelling evidence for a network of activity of pluripotency and stem-associated genes comprising of Oct4, Nanog and nestin. During neurogenesis, the choice between enhancement versus suppression of transcriptional modulation of these identified genes determines the balance between self-renewal neural stem cells (NSC) and immature neuronal phenotypes. By using immunocytochemistry and RT-PCR techniques, our study aims to address the question whether and to what extent mRNA and protein profiles are expressed in human fetal neurospheres obtained from cortical and striatal brain regions, both in expansion (undifferentiated cells) and differentiation conditions monitored after 1 and 4 weeks in vitro culturing. Our results clearly demonstrate the sustained presence of opposite signals: strong downregulation of Oct4 and Nanog genes in cortical differentiating cells and significant up-regulation for nestin gene both in cortical and striatal differentiating cells. Notably, by immunostaining techniques, Oct4 and Nanog protein expression have indicated the presence of both nuclear and cytoplasmic content followed by their rapid turnover (immediately after 1 week). Moreover, during the differentiation process, dissociated neurospheres displayed unexpected number of nestin positive cells accompanied by a constant level of staining intensity. In conclusion, the present study provides new insights into brain region related features in terms of Oct4, Nanog and nestin expression both at cellular and molecular level.

Spontaneous Transformation of Stem Cells In Vitro and the Issue of Cross-Contamination

We have read with interest the paper “Long-term cultured human neural stem cells undergo spontaneous transformation to tumor-initiating cells”, recently published by Wu et al. 1. In this study the authors show spontaneous transformation of human fetal striatum neural stem cells (hsNSCs) in culture, and that the transformed cells (T1) are characterized by stem cell-like features, the expression of neural stem cell markers, abnormal karyotype and an increased growth rate. In the text they refer to previous reports on spontaneous MSC transformation 2, 3. However, they fail to inform the readers that both these publications have later been retracted or corrected since both research groups detected that their transformed cells were cross-contaminated with cancer cells 4, 5. In the article by Wu and colleagues, they have characterized the T1 cells by DNA fingerprinting. Most interesting, the DNA fingerprint of the transformed cells (T1) did not match the “cell of origin”, and the authors explain this by genetic instability. However, we have compared the T1 fingerprint published by Wu et al., with public available cell line STR profiles, and find that the T1 STR profile published by Wu et al. is surprisingly similar to HeLa cells, Table ​Table1.1. The DNA fingerprinting profile of cancer cells compared to normal cells is characterized by large differences in peak height at one or more loci, indicating genetic instability, occasional additional alleles at a locus, indicating gene duplication events, and loss of heterozygosity (LOH), at one or more loci 6, 7. Genetic imbalance will in other words not generate a completely new fingerprinting profile. Table 1 STR fingerprinting profile of hsNSC, T1 and HeLa STR profiling is currently the recommended test for cell line authentication due to its high power of discrimination and the possibility to compare the numerical code obtained from various laboratories 7. Wu and colleagues analyzed their cells by using the PowerPlex 16 System Kit from Promega. The kit provides 15 STR markers as well as the gender determinator Amelogenin, and it has a matching probability of > 1 in 1.83×10e17 (www.promega.com). The same kit was recently used to determine the STR profile of HeLa cells 8, showing 97% identity between T1 and HeLa with only one LOH (Table ​(Table1).1). According to general recommendations, the profile of identical or closely related profiles should match at 80% or more of the alleles 6, and profiles with an identity level between 50 and 75% must be regarded with suspicion 7. The HeLa profiles listed in Table ​Table11 match T1 with 80-97% accuracy. A minor variation is seen at one locus when comparing the STR profile reported by ATCC and CLS (D13S317: 12,13.3 and 13,13.3) and Wu (D13S317: 12,14). According to the Promega Protocol for PowerPlex16, each allele at the D13S317 locus separates by 4 nucleotides, and it is therefore unclear if the allele at D13S317 13.3 is correct. There is at present several batches of HeLa cells available, and minor differences exists between them 6. A number of scientists have pointed at the problem of cross-contamination for decades, and it is now highly recommended to authenticate cells by DNA fingerprinting 7, 9. Several databases for checking the fingerprinted profiles are available, such as STR profile databases at ATCC (www.lgcstandards-atcc.org) and DSMZ (www2.dsmz.de). Also a list of 360 cross-contaminated cell lines is available to help researchers quality-check their work 10, and HeLa is still the most frequent cross-contaminating cell line 10. In conclusion, it is highly questionable if the article presented by Wu et al., actually describes a transforming event of hsNSCs.