Havard Olstorn

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.

Transplantation of stem cells from the adult human brain to the adult rat brain.

OBJECTIVETo investigate the migration, proliferation, and differentiation of stem cells and neural progenitor cells (NPCs) from the adult human brain after transplantation into adult rodent brains. METHODSAdult human NPCs were obtained from temporal lobe specimens removed because of medical intractable epilepsy. The cells were transplanted into the posterior periventricular region above the hippocampus in the brains of either healthy adult rats (control) or rats with selective injury of the hippocampal CA1 region (global ischemia). RESULTSIn the control animals, grafted cells were mainly distributed from the site of transplantation toward the midline along white matter tracts. The density of transplanted cells elsewhere, including the hippocampus, was low and apparently random. In animals with CA1 damage, NPCs showed targeted migration into the injured area. Cell survival at 10 weeks was 4.7 ± 0.3% (control, n = 3) and 3.7 ± 1.1% (ischemia, n = 3); at 16 weeks, cell survival was 3.4 ± 0.6% (control, n = 2) and 7.2 ± 1.5% (ischemia, n = 2), i.e., comparable to what has been observed earlier when transplanting embryonic tissue into the human brain or progenitor cells between inbred rats. The number of dividing cells decreased with time. Sixteen weeks after transplantation, 4 ± 1% (n = 4) of the cells showed proliferative activity. We did not observe signs of tumor formation or aberrant cell morphology. Neuronal differentiation was much slower than what has been observed earlier in vitro or after transplantation to the developing nervous system, and 16 weeks after transplantation many surviving cells were still in maturation. CONCLUSIONThe present study shows that adult human NPCs survive, show targeted migration, proliferate, and differentiate after grafting into the adult rat brain.

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.

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.

Neurogenesis and Potential Use of Stem Cells from Adult Human Brain

Neural stem cells are present in the adult human brain of mammals, including humans, and can give rise to the three major cell types of the central nervous system; neurons, astrocytes and oligodendrocytes. These stem cells hold great promise for neural repair after injury or disease, either by activating the stem cells residing within the brain and/or by transplantation of stem cells from the adult human brain after expanding them in culture dishes. Autologous transplantation, in which a patient is transplanted with cells derived from his or her own brain, could circumvent some of the problems associated with the use of embryonic stem cells or fetal tissue, in particular the ethical concerns and problems with immune rejection. However, it must be demonstrated that the necessary types of neural cells can be generated in sufficient amounts, and that they can induce long-lasting functional improvements in animal models of brain disease and injury.