Mercy Varghese

Stem cells from the adult human brain develop into functional neurons in culture.

Recent research communications indicate that the adult human brain contains undifferentiated, multipotent precursors or neural stem cells. It is not known, however, whether these cells can develop into fully functional neurons. We cultured cells from the adult human ventricular wall as neurospheres and passed them at the individual cell level to secondary neurospheres. Following dissociation and plating, the cells developed the antigen profile of the three main cell types in the brain (GFAP, astrocytes; O2, oligodendrocytes; and beta-III-tubulin/NeuN, neurons). More importantly, the cells developed the electrophysiological profiles of neurons and glia. Over a period of 3 weeks, neuron-like cells went through the same phases as neurons do during development in vivo, including up-regulation of inward Na+ -currents, drop in input resistance, shortening of the action potential, and hyperpolarization of the cell membrane. The cells developed overshooting action potentials with a mature configuration. Recordings in voltage-clamp mode displayed both the fast inactivating TTX-sensitive sodium current (INa) underlying the rising phase of the action potential and the two potassium currents terminating the action potential in mature neurons (IA and IK, sensitive to 4-AP and TEA, respectively). We have thus demonstrated that the human ventricular wall contains multipotent cells that can differentiate into functionally mature neurons.

A comparison between stem cells from the adult human brain and from brain tumors.

OBJECTIVE To directly compare stem cells from the normal adult human brain (adult human neural stem cells [AHNSC]), Grade II astrocytomas (AC II), and glioblastoma multiforme (GBM), with respect to proliferative and tumor-forming capacity and differentiation potential. METHODS Cells were isolated from tissue obtained during epilepsy surgery (AHNSCs) or tumor surgery (glioma stem cells [GSC]). They were cultured and investigated in vitro or after transplantation in immunodeficient mice. RESULTS Under identical experimental conditions, the following were found: 1) GBM stem cells formed tumors after orthotopic transplantation; AHNSCs showed no sign of tumor formation; 2) GSCs showed a significantly higher growth rate and self-renewal capacity; 3) both the growth rate and telomerase expression were high in GSCs and correlated with malignancy grade (GBM higher than AC II); AHNSCs had low telomerase expression; 4) GSCs invaded normal neurospheres, not vice versa; 5) both AHNSCs and stem cells from AC II and GBM responded to differentiation cues with a dramatic decrease in the proliferation index (Ki-67); 6) GSCs differentiated faster than AHNSCs; 7) upon differentiation, AHNSCs produced normal glia and neurons; GSCs produced morphologically aberrant cells often expressing both glial and neuronal antigens; and 8) differentiation of AHNSCs resulted in 2 typical functional phenotypes: neurons (high electrical membrane resistance, ability to generate action potentials) and glial cells (low membrane resistance, no action potentials). In contrast, GSCs resulted in only 1 functional phenotype: cells with high electrical resistance and active membrane properties capable of generating action potentials. CONCLUSION AHNSCs and stem cells from AC II and GBM differ with respect to proliferation, tumor-forming capacity, and rate and pattern of differentiation.

Development of neuronal networks from single stem cells harvested from the adult human brain.

OBJECTIVE: It was long held as an axiom that new neurons are not produced in the adult human brain. More recent studies, however, have identified multipotent cells whose progeny express glial or neuronal markers. This discovery may lead to new therapeutic strategies against central nervous system disorders by transplanting stem cells that have been propagated in vitro. Still, it is not known whether stem cells from the adult human brain retain the potential to mature into neurons that integrate and communicate in a network. METHODS: We cultured cells from the ventricular wall of the adult human brain as monoclonal neurospheres. After two passages, the neurospheres were dissociated and the cells were allowed to differentiate. After 4 weeks of maturation, the cells were studied by immunocytochemistry, confocal microscopy, and whole-cell patch-clamp. RESULTS: We show that monoclonal stem cells harvested from the ventricular wall of the adult human brain develop into mature neurons with functional glutamate receptors and glutamatergic nerve terminals. By patching pairs of cells simultaneously, we also present direct evidence for synaptic communication between neurons developed from the same monoclonal cell. CONCLUSION: Neural stem cells harvested from the adult human brain retain the potential to mature into fully differentiated neurons that integrate and communicate by synapses. This opens a possible future scenario of autotransplantation, in which stem cells are harvested from small biopsies of the ventricular wall and propagated in vitro before transplantation.

A comparison of epithelial and neural properties in progenitor cells derived from the adult human ciliary body and brain.

Cells isolated from the ciliary body (CB) of the adult human eye possess properties of retinal stem/progenitor cells and can be propagated as spheres in culture. As these cells are isolated from a non-neural epithelium which has neuroepithelial origin, they may have both epithelial and neural lineages. Since it is the properties of neural progenitor cells that are sought after in a future scenario of autotransplantation, we wanted to directly compare human CB spheres with neurospheres derived from the human subventricular zone (SVZ), which is the best characterized neural stem cell niche in the CNS of adults. The CB epithelium was dissected from donor eyes (n = 8). Biopsies from the ventricular wall were harvested during neurosurgery due to epilepsy (n = 7). CB and SVZ tissue were also isolated from Brown Norwegian rats. Dissociated single cells were cultivated in a sphere-promoting medium and passaged every 10-30 days. Fixed spheres were studied by immunohistochemistry, quantitative RT-PCR and scanning/transmission electron microscopy. We found that both CB and SVZ spheres contained a mixed population of cells embedded in extracellular matrix. CB spheres, in contrast to SVZ neurospheres, contained pigmented cells with epithelial morphology that stained for cytokeratins (3/12 + 19), were connected through desmosomes and tight-junctions and produced PEDF. Markers of neural progenitors (nestin, Sox-2, GFAP) were significantly lower expressed in human CB compared to SVZ spheres, and nestin positive cells in the CB spheres also contained pigment. There was higher expression of EGF and TGF-beta receptors in human CB spheres, and a comparative greater activation of the canonical Wnt pathway. These results indicate that adult human CB spheres contain progenitor cells with epithelial properties and limited expression of neural progenitor markers compared to CNS neurospheres. Further studies mapping the regulation between epithelial and neural properties in the adult human CB spheres are vital to fully utilize them as a clinical source of retinal progenitor cells in the future.

Brain tumor stem cells maintain overall phenotype and tumorigenicity after in vitro culturing in serum-free conditions

Traditional in vitro culturing of tumor cells has been shown to induce changes so that cultures no longer represent the tumor of origin. Serum-free culturing conditions are used in a variety of cancers to propagate stem-like cells in vitro. Limited reports, however, exist on the effects of such propagation. We have compared cells from brain tumor biopsies cultivated under serum-free conditions at passages 2 and 10 to describe the effects of in vitro culturing. We were able to establish cell lines from 7 of 10 biopsies from patients with glioblastoma. The cell lines adapted to conditions and had 2.2 times increased population doubling rate at later passages. Karyotyping and comparative genomic hybridization analysis revealed that all examined cell lines had cytogenetic aberrations commonly found in glioblastomas, and there were only minor differences between tumor and early and late passages in the same culture. Whole-transcriptome analysis shows that tumors had interindividual differences. Changes in the overall expression patterns through passaging were modest, with a significant change in only 14 genes; the variation among cultures was, however, reduced through passages. The ability to differentiate differed among tumors but was maintained throughout passaging. The cells initiated tumors upon transplantation to immunodeficient mice with differing phenotypes, but a given cell culture maintained tumor phenotype after serial cultivation. The cultures established maintained individual characteristics specific to culture identity. Thus, each cell culture reflects an image of the tumor--or a personalized model--from which it was derived and remains representative after moderate expansion.

Artificial Niches for Human Adult Neural Stem Cells: Possibility for Autologous Transplantation Therapy

Cellular transplantation therapy is thought to play a central role in the concept of restorative neurosurgery, which aims to restore function to the damaged nervous system. Stem cells represent a potentially renewable source of transplantable cells. However, control of the behavior of these cells, both in the process of clonogenic expansion and post-transplantation, represents formidable challenges. Stem cell behavior is thought to be directed by extracellular signals in their in vivo niches, many of which are protein or peptide based. As only one example, activation of Notch plays an important role in normal development and is the strongest known signal for stem cells to choose glial over neuronal fates. Therefore, artificial extracellular matrix proteins represent a potentially powerful tool to custom design artificial niches to strategically control stem cell behavior. We have developed a family of aECM proteins that incorporate the active domains of the DSL ligands to the Notch receptor into an elastin-based backbone. The development of our DSL-elastin artificial proteins demonstrates the design strategy and methodology for the production of bioactive artificial extracellular matrix proteins aimed at modulating stem cell behavior, and this method can be used to design other bioactive aECM proteins. In addition, we have developed a method for the isolation and characterization of adult human neural stem cells from periventricular tissue harvested from living patients. This paper reviews cellular transplantation therapy from the clinical perspective and summarizes ongoing work aimed at exploring the intriguing possibility of autologous transplantation, whereby neural stem cells can be harvested from adult patients, expanded or modified in vitro in artificial niches, and retransplanted into the original patient.

Aqp 9 and brain tumour stem cells.

Several studies have implicated the aquaporins (aqp) 1, 4, and 9 in the pathogenesis of malignant brain tumours, suggesting that they contribute to motility, invasiveness, and oedema formation and facilitate metabolism in tumour cells under hypoxic conditions. We have studied the expression of aqp1, 4, and 9 in biopsies from glioblastomas, isolated tumour stem cells grown in a tumoursphere assay and analyzed the progenitor and differentiated cells from these cultures. We have compared these to the situation in normal rat brain, its stem cells, and differentiated cells derived thereof. In short, qPCR in tumour tissue showed presence of aqp1, 4, and 9. In the tumour progenitor population, aqp9 was markedly more highly expressed, whilst in tumour-derived differentiated cells, aqp4 was downregulated. However, immunostaining did not reveal increased protein expression of aqp9 in the tumourspheres containing progenitor cells; in contrast, its expression (both mRNA and protein) was high in differentiated cultures. We, therefore, propose that aquaporin 9 may have a central role in the tumorigenesis of glioblastoma.

Isolation of Human Multipotent Neural Progenitors from Adult Filum Terminale

Stem cells have been isolated from several CNS regions, including the spinal cord. However, the terminal end of the spinal cord, filum terminale, has been referred to as a fibrovascular tag without neurogenic potential and of no clinical significance. Recently, we were fortunate to acquire some samples of this tissue. We show for the first time that progenitor cells exhibiting the hallmarks of stem cells can be isolated from adult human filum terminale (FTNPs). More specifically, FTNPs self-renew and proliferate to form neurospheres, and exhibit tripotent differentiation into neurons, astrocytes, and oligodendrocytes. Equally important, FTNPs develop the electrophysiological profile of neurons and glia. Whole-cell patch-clamp recordings show beta-III-tubulin(+) neurons exhibiting overshooting action potentials, displaying both the fast inactivating TTX-sensitive sodium current as well as 4-AP and TEA sensitive potassium currents. To assess potency in vivo, FTNPs were transplanted into the posterior periventricular region of control or ischemic rat brains. Despite a vigorous immune response against the xenograft, FTNPs survived and were found not only in the graft area but had also migrated to the lesioned CA1 region. Notwithstanding the immune response, FTNPs differentiated into astrocytes, but no neuronal differentiation was observed in the transplant milieu tested. However, neuronal differentiation in vivo cannot be ruled out and assessment of the conditions necessary to promote neurogenesis in vivo requires more research. Significantly, no tumor formation or aberrant cell morphology was seen in or adjacent to the graft area. Thus, filum terminale provides a novel source of adult human neural progenitor cells that develop into functional neurons with possible clinical applications.

Predifferentiated Brain-Derived Adult Human Progenitor Cells Migrate Toward Ischemia After Transplantation to the Adult Rat Brain

BACKGROUND:The adult human brain contains neural stem/progenitor cells (AHNPCs) that can survive transplantation into the adult rat brain, migrate toward a lesion, and display limited neuronal differentiation in vivo. OBJECTIVE:To investigate the effect of manipulating AHNPCs before grafting by predifferentiation, ie, initiating neuronal differentiation before transplantation, and to determine whether this cell priming would affect their ability to migrate in vivo. METHODS:AHNPCs were prepared from temporal lobe resections for epilepsy. Seven days after global brain ischemia, predifferentiated AHNPCs (exposed to basic fibroblast growth factor, heparin, and laminin) were transplanted to the left hippocampus. Four and 10 weeks after transplantation, brain sections were analyzed by immunohistochemistry. RESULTS:Transplanted primed cells expressed committed neuronal markers at a much earlier stage compared with nonprimed AHNPCs and were found colabeled with human markers within the damaged CA1 region 4 weeks after grafting. Furthermore, predifferentiated AHNPCs migrated preferentially into an ischemic lesion, similar to their undifferentiated counterparts. The chemoattractant effect from the expression of stromal cell–derived factor-1α (SDF-1α) in ischemic CA1 on AHNPCs expressing CXC chemokine receptor 4 (CXCR4) may explain this preference in migration in vivo. CONCLUSION:The plasticity of neural progenitors derived from the adult human brain may be greater than previously assumed in that manipulation before grafting may influence the phenotypes seen in vivo. The SDF-1α–CXCR4 axis is involved in the targeted migration toward an ischemic lesion in the adult rat brain, similar to previous reports on endogenous progenitors in rats and grafted fetal human neural progenitors.

Symptomatic spinal metastasis from intracranial glioblastoma multiforme - Two illustrative cases and a review of the literature.

Glioblastoma multiforme (GBM) was first described in 1863 [1] and is the most aggressive primary malignancy of the central nervous system (CNS) [2]. The incidence in Europe is 2–3 cases per 100 000 individuals per year and the median age at diagnosis is approximately 60 years [2]. GBM is a highly proliferative tumor and displays extensive invasion of normal brain tissue causing inevitable tumor recurrence, resulting in one of the worst fiveyear survival rates among all human cancers [3]. In fact, about 75% of patients die within 18 months after GBM diagnosis [2]. Metastasis from GBM via the cerebrospinal fluid (CSF) to the spinal compartment was first described by Cairns and Russell in 1931 [4]. Notwithstanding the relative paucity of this phenomenon in the literature, spinal dissemination of GBM has been reported with increasing frequency [5,6]. In fact, CSF dissemination is said to occur in about 20% of supratentorial cases of GBM [5] and in about 60% of infratentorial cases. Interestingly, autopsy series have shown a higher incidence of GBM metastasis to the spinal compartment than clinically observed [7], suggesting that such metastases are usually asymptomatic and thus underdiagnosed to date. Treatment is palliative and prognosis is very poor, with the average time interval between diagnosis of metastatic disease and death being 2–3 months [8]. Although the reason for the increased frequency of reported cases of spinal dissemination of GBM remains controversial, factors that play a significant role include advances in imaging of the neuroaxis and aggressive neurooncological treatment [5]. Such advances have improved local tumor control leading to prolonged survival and, hence, increased the probability of metastatic spread as well as symptomatic manifestations of such metastases. Improvements in surgical and neurooncological management of GBM are inevitable and will most likely lead to an increase in the frequency of clinically relevant spinal metastases. We present two patients who developed symptomatic spinal metastasis after undergoing multimodal neurooncological treatment for GBM. Increased awareness and clinical suspicion of this complication is necessary in GBM patients presenting with back pain or symptoms of spinal cord compression, as this may lead to earlier diagnosis and management of metastatic disease. The research on this case report was conducted in accordance with the Declaration of Helsinki and in accordance with the guidelines at The Norwegian Radium Hospital. Case reports