TY - JOUR
AU - Parati, Eugenio A.
AB - Abstract We have recently isolated stem cells deriving from the olfactory bulbs of adult patients undergoing particularly invasive neurosurgery. After improving our experimental conditions, we have now obtained neural stem cells according to clonal analysis. The cells can be expanded, established in continuous cell lines and differentiated into the three classical neuronal phenotypes (neurons, astrocytes, and oligodendrocytes). Also, after exposition to leukemia inhibitory factor, we are able to improve the number of neurons, an ideal biological source for transplantation in various neurodegenerative disorders. Neural stem cell, Human adult olfactory bulb, Transplantation, Parkinson's disease, Multipotent precursor, Cell migration Introduction The olfactory lobe arises from the rostral-ventral portion of the telencephalic vesicle, and can be divided into two principal parts: a posterior part including the lobe of the hippocampus and other structures, and an anterior part including the tubercle, peduncle, and olfactory bulb. Widespread cell migrations are the hallmark of vertebrate brain development. In the early embryo, morphogenetic movements of precursor cells establish the rhombomeres of the hindbrain and the region bound by the forebrain. In mid-gestation, individual postmitotic cells undergo directed migrations along the glial fiber system, and radial migration establishes the neuronal layers. In the postnatal period, a wave of secondary neurogenesis produces large numbers of interneurons destined for the cerebral cortex and olfactory bulb [1, 2]. De novo neurogenesis has been reported in the subgranular region of the dentate gyrus, the cortex [3, 4], and olfactory bulb of various adult rodent brains [5-7]. In 1992, Reynolds and Weiss reported the presence of subventricular zone (SVZ) neural stem cells during rodent brain development [8]. Neural stem cells can only be characterized on a critical functional basis in terms of their undifferentiated features, capacity for self-renewal, pluripotentiality, and ability to regenerate damaged tissue [9]. Neural stem cells with these characteristics have been found in cultures of embryonic and adult murine brain [10-14], as well as in human embryonic brain explants after epigenetic stimulation [15, 16]. The SVZ is an intensive proliferative zone and the progeny of neural stem cells can either die or give rise to “neural progenitors” that migrate to the olfactory bulb [17]. We have for the first time isolated stem cells from the olfactory bulbs of adult patients undergoing particularly invasive neurosurgery. Under our optimized conditions, stem cells isolated from the olfactory bulb proliferate in culture in the same way as human embryonic stem cells after epigenetic stimulation and retain all of the typical characteristics of neural stem cells: like human embryonic cells, they proliferate in response to mitotic factors (basic fibroblast growth factor, bFGF, and epidermal growth factor, EGF) and have the ability to give rise to neurons, astrocytes, and oligodendrocytes [15]. The novel aspects of this discovery are the combination of the intense regeneration potential of the olfactory bulb and the possibility of explanting autologous neural stem cells from patients affected by different neurodegenerative disorders by means of simple partial bulbectomy. Finally, as a result of our many years of experience in manipulating these cells, we are able to induce their expansion in vitro and improve their differentiation in neurons, thus representing the ideal biological source for autotransplantation in these patients. Material and Methods Isolation, Expansion, and Multipotentiality of Human Adult Olfactory Bulb Stem Cells: Establishment of Cell Lines. The tissue was obtained following the ethical guidelines of the European Network for Transplantation and the Declaration of Helsinki from patients undergoing particularly invasive neurosurgery. Olfactory bulb tissue was placed in artificial colony-stimulating factor ([aCSF] 124 mM NaCl, 5 mM KCl, 1.3 mg Cl2, 0.1 mM CaCl2, 26 mM NaHCO3, and 10 mM D-glucose, pH 7.3) aerated with 95% O2/5% CO2 at room temperature. The tissue was dissociated and transferred into 15 ml of CSF containing 1.3 mg/ml trypsin (type XII, 9,000 BASF unit/mg; Sigma; St. Louis, MO; http://www.sigma-aldrich.com), 0.67 mg/ml hyarulonidase (2,000 units/mg; Sigma), and 0.2 mg/ml kynurenic acid (Sigma) and incubated under continuos oxygenation and stirring for 90 min at 32-34°C. Tissues were then rinsed in aCSF for 10 min, transferred to Dulbecco's modified essential medium (DMEM)/F12 (1:1 v/v; Life Technologies; Gaithersburg, MD; http://www.lifetech.com), containing 0.7 mg/ml ovomucoid (Sigma), and carefully triturated with a fire-polished Pasteur pipette. The cell suspension was collected by centrifugation and plated at 500 cells/cm2 in untreated 25 cm2-tissue culture flasks (Nunc; Naperville, IL; http://www.nalgenunc.com) in the presence of, respectively, 20 ng/ml and 10 ng/ml of human recombinant EGF and bFGF in basal serum-free medium (a DMEM-F12 medium whose nutrient composition is optimized for neural stem cell growth; Euroclone; Irvine, Scotland) containing 2 mM L-glutamine, 0.6% glucose, 9.6 g/ml putrescine, 6.3 ng/ml progesterone, 5.2 ng/ml sodium selenite, 0.025 mg/ml insulin, and 0.1 mg/ml transferrin (sodium salt, grade II; Sigma). Two weeks later, the cultures were harvested, mechanically dissociated and replated under the same conditions. After performing this procedure twice in order to eliminate short-term dividing precursors, bulk cultures were generated by passing the cells at a higher density (104 cells/cm2) in the same growth medium every 10 days. Cell counts and viability tests were performed at every passage by means of trypan blue exclusion (as previously reported) [14]. In order to demonstrate multipotentiality after differentiation, the stem cell progeny were plated at very low density onto laminine-coated (Boehringer Mannhneim; Indianapolis, IN) chamber slides (Labtek®; NUNK; Naperville, IL; http://www.labtek.net) in DMEM/F12 medium with 2% fetal calf serum for four days before immunocytochemical analysis. Improving Adult Human Olfactory Bulb Stem Cell-Derived Neuronal Progeny Using Leukemia Inhibitory Factor (LIF) The stem cell progenies obtained from olfactory bulb explantation were plated at 500 cells/cm2 onto laminine-coated (Boehringer Mannhneim) chamber slides (Labtek®) in mitotic factor and serum-free DMEM/F12 medium containing LIF (20 ng/ml) for 15 days. After differentiation, the neurons were counted every five days and the result compared with the total cell number. Immunocytochemistry The cell composition was analyzed by means of immunostaining with lineage-specific antibodies. The cells were fixed for 20 min in 4% paraformaldehyde in phosphate-buffered saline (PBS), pH 7.4, and then washed and incubated with PBS/0.1% Triton-X containing 10% normal goat serum and the appropriate antibody mixture for 90 min at 37°C. The primary antibodies were rabbit anti-Glial fibrillary acidic protein (ready to use; Incstar Corporation; Stillwater, MN), mouse anti-β-tubulinIII (1:1000; Sigma) and anti-Galactocerebroside (1:300; Sigma). After some washing, the cultures were incubated for 45 min at room temperature with secondary fluorescein isothiocyanate- or rhodamine isothiocyanate-conjugate goat antimouse or antirabbit IgG antibodies (1:100; Boehringer Mannheim), washed, incubated with 4′, 6-diamidino2-phenylinole dihydrochcloride (1 mg/ml in methanol, 15 min at 37°C [14]), and finally mounted using Fluorsave™ (Calbiochem; La Jolla, CA; http://www.calbiochem.com) and viewed under a Zeiss (Oberkochen, Germany; http://www.zeiss.com) Axiophot-2 microscope. Results Isolation and Expansion of Human Adult Olfactory Bulb Stem Cells: Establishment of Cell Lines. Neural stem cells have been found in cultures of embryonic and adult murine brain, as well as embryonic human brain diencephalon, after epigenetic stimulation [13-16]. We have for the first time established the conditions for isolating and propagating neural stem cells from the olfactory bulb of adult human subjects. This was a difficult process because of the intrinsic characteristics of the cells (the small number of stem cells and degraded state of the tissue) and the small number of vital cells obtained from a single adult subject. The individual cells were then plated in growth medium containing 20 ng/ml of EGF and 10 ng/ml of fibroblast growth factor (FGF). When exposed to these mitotic factors, approximately 1% of the cells are “real” stem cells, 70% died, and the others are transiently proliferating progenitors, mostly stuck on the flask surface. Early daughter cells undergo transient proliferation, followed by cell differentiation after a few weeks and eventual death. Conversely, in these condition, the surviving floating cells began to divide and gave rise to spherical clusters which were composed of undifferentiated neural precursor, as demonstrated by the lack of any specific morphology, the absence of neural and glial antigens, and the expression of central nervous system precursor marker nestin (not shown) [15]. Self-renewal was demonstrated by serial subcloning experiments. Single spheres were dissociated and replated in growth medium at low density (less than 100 cells/cm2) to prevent cell aggregation. When individual primary spheres were dissociated and replated as single cells under clonal condition in growth medium, some cells either differentiated to acquire the typical morphology of neuronal/glial cells or died. However, a subset of cells proliferated and gave rise to an average of 6 ± 2 secondary spheres (approximately 150 μm of diameter). Thus the number of 2° neurospheres is equal to the neural stem cells in the original culture [14, 15]. The process is maintained constantly even at late passages in vitro (not shown). In order to amplify the neural stem cells number, the floating neurospheres were isolated by low-speed centrifugation and then plated in untreated 25 cm2 tissue culture flasks containing growth medium. The entire process was repeated four times, with the cultures being mechanically dissociated to single-cell suspensions every five days and then replated in fresh growth medium. Using this approach, the cultures proliferated efficiently giving rise to “olfactory bulb stem cell lines.” At each passage, the culture expansions were monitored using the trypan-blue method. As shown in Figure 1, the number of olfactory bulb neural stem cells increased significantly after 15 days in vitro (DIV) and the proliferation rate became exponentially higher after about 20 DIV. Observation of single proliferating cells revealed that the first division always occurred after 3-4 DIV in cells from either early fifth or late fifteenth passages. Accordingly, the slope of the stem cells growth curve remained constant even after a significant number of serial passages. Giving the slow rate of proliferation of adult olfactory bulb neural stem cells, we have successfully maintained cell lines in culture until twentieth passages in vitro. They are cryopreservable and retain full proliferation and differentiation capacity even in long-term cultures (Table 1) [14, 15]. As already reported in other literature, our cell lines possess the critical stem characteristics such as: A) undifferentiated features (as defined by the lack of differentiated markers); B) self-renewing capacity, and C) pluripotentiality [9, 18], so they can really be defined as olfactory bulb neural stem cells [19]. Thus, likewise in embryonic stem cells, the adult olfactory bulb derived stem cells can be amplified in vitro like embryonic neural stem cells, and it is possible to significantly increase their small starting number. Figure 1. Open in new tabDownload slide Growth curve of human olfactory bulb stem cells during the first four weeks. After a period of relatively slow growth (time of doubling between 5-10 DIV is 5 days), the cells quickly expand (time of doubling 15-20 DIV is 2.5 days). The cells were cultured in a growth medium containing mitotic factors (20 ng/ml of EGF and 10 ng/ml of bFGF). Figure 1. Open in new tabDownload slide Growth curve of human olfactory bulb stem cells during the first four weeks. After a period of relatively slow growth (time of doubling between 5-10 DIV is 5 days), the cells quickly expand (time of doubling 15-20 DIV is 2.5 days). The cells were cultured in a growth medium containing mitotic factors (20 ng/ml of EGF and 10 ng/ml of bFGF). Table 1. Maintenance of multipotentially in early and late cell lines . Neurons . Astrocytes . Oligodendrocytes . 7 DIV 16.21 ± 1.49% 65.8 ± 2.6% 2.1 ± 0.96% (3 passages) 40 DIV 13.68 ± 0.68% 70.21 ± 1.38% 3.2 ± 1.98% (15 passages) . Neurons . Astrocytes . Oligodendrocytes . 7 DIV 16.21 ± 1.49% 65.8 ± 2.6% 2.1 ± 0.96% (3 passages) 40 DIV 13.68 ± 0.68% 70.21 ± 1.38% 3.2 ± 1.98% (15 passages) Data are the mean ± SE of four independent experiments. Open in new tab Table 1. Maintenance of multipotentially in early and late cell lines . Neurons . Astrocytes . Oligodendrocytes . 7 DIV 16.21 ± 1.49% 65.8 ± 2.6% 2.1 ± 0.96% (3 passages) 40 DIV 13.68 ± 0.68% 70.21 ± 1.38% 3.2 ± 1.98% (15 passages) . Neurons . Astrocytes . Oligodendrocytes . 7 DIV 16.21 ± 1.49% 65.8 ± 2.6% 2.1 ± 0.96% (3 passages) 40 DIV 13.68 ± 0.68% 70.21 ± 1.38% 3.2 ± 1.98% (15 passages) Data are the mean ± SE of four independent experiments. Open in new tab Demonstration of the Multipotentiality of Adult Human Olfactory Bulb Neural Stem Cells The multipotentiality of stem cells is demonstrated by their capacity to generate neurons, oligodendrocytes, and astrocytes. As only clonal analysis proved multipotentiality, we cultured the neural bulb stem cells under proliferating conditions at low density (Figs. 2A, 2B and 2C), and we are able to differentiate the olfactory single spheres into the three classical neuronal phenotypes. Figure 2. Open in new tabDownload slide Single cells derived from the dissociation of neurospheres were plated into a laminine-coated substrate. The grey sign on the right identifies the field (arrowhead). The central cell of the field, after the removal of the mitotic factors, proliferated and gave rise to a clonal progeny; subsequently, the progeny began to expand and differentiate (Fig. 2A and 2B and 2C). The culture was finally processed for immunofluorescence assay. Bar: 12 μm Figure 2. Open in new tabDownload slide Single cells derived from the dissociation of neurospheres were plated into a laminine-coated substrate. The grey sign on the right identifies the field (arrowhead). The central cell of the field, after the removal of the mitotic factors, proliferated and gave rise to a clonal progeny; subsequently, the progeny began to expand and differentiate (Fig. 2A and 2B and 2C). The culture was finally processed for immunofluorescence assay. Bar: 12 μm The progeny obtained from the central single cell in the field, indicated with a gray sign (arrowhead, Fig. 2A), were found to give rise to cells expressing neuronal, astroglial, and oligodendroglial antigens (Fig.3A, a neuron immunoreactive to β-tubulinIII; Fig.3B, an oligodendrocyte immunoreactive to GalC; Fig. 3C, an astrocyte immunoreactive to GFAP). It is worth noting that the neuronal differentiation occurred gradually and neuronal processes progressively elongated over time. These findings together unequivocally demonstrate the multipotentiality of these neural stem cells. Furthermore, it has previously been shown that these neurons have excitable properties [15]. Figure 3. Open in new tabDownload slide Multiple immunolabeling of the progeny obtained from the single cell previously indicated in Figure 2A shows the presence of the all-neural phenotypes, thus demonstrating the full multipotential of the olfactory bulb stem cells. Figure 3A shows a neuron immunoreactive to β-tubulinIII; Figure 3B shows an oligodendrocyte immunoreactive to GalC; Figure 3C shows an astrocyte immunoreactive to GFAP. Bar: 12 μm Figure 3. Open in new tabDownload slide Multiple immunolabeling of the progeny obtained from the single cell previously indicated in Figure 2A shows the presence of the all-neural phenotypes, thus demonstrating the full multipotential of the olfactory bulb stem cells. Figure 3A shows a neuron immunoreactive to β-tubulinIII; Figure 3B shows an oligodendrocyte immunoreactive to GalC; Figure 3C shows an astrocyte immunoreactive to GFAP. Bar: 12 μm LIF Increases the Number of Olfactory Bulb Neural Stem Cell-Generated Neurons As already reported, exposition to LIF does not alter stem cell proliferation and/or number, while LIF influences the survival during the differentiation process [16]. In agreement with the significant expression of the transcript for the LIF-β-receptor in human stem cell cultures [16], exposure to LIF had a striking effect on neuronal differentiation. In comparison with control cultures, there was a twofold increase in the number of cells immunolabeled with β-tubulinIII after only five days of exposure to LIF, and this difference was maintained after ten and fifteen days in vitro (Table 2). Table 2. Percentage of β-tubulin III immunoreactivity/total number of cells . 5 DIV . 10 DIV . 15 DIV . Growth culture medium without EGF and bFGF 6.13 ± 1.88 9.66 ± 1.37 16.66 ± 2.95 Growth culture medium without EGF and bFGF with LIF (20 ng/ml) 11.12 ± 2.43 18.13 ± 1.99 35. 06 ± 2.63 . 5 DIV . 10 DIV . 15 DIV . Growth culture medium without EGF and bFGF 6.13 ± 1.88 9.66 ± 1.37 16.66 ± 2.95 Growth culture medium without EGF and bFGF with LIF (20 ng/ml) 11.12 ± 2.43 18.13 ± 1.99 35. 06 ± 2.63 Data are the mean ± SE of two independent experiments. Each increment was statistically significant at p < 0.005 (Student t-test); n = 4. DIV = days in vitro; LIF = leukemia inhibitory factor; EGF = epidermal growth factor; bFGF = basic fibroblast growth factor. Open in new tab Table 2. Percentage of β-tubulin III immunoreactivity/total number of cells . 5 DIV . 10 DIV . 15 DIV . Growth culture medium without EGF and bFGF 6.13 ± 1.88 9.66 ± 1.37 16.66 ± 2.95 Growth culture medium without EGF and bFGF with LIF (20 ng/ml) 11.12 ± 2.43 18.13 ± 1.99 35. 06 ± 2.63 . 5 DIV . 10 DIV . 15 DIV . Growth culture medium without EGF and bFGF 6.13 ± 1.88 9.66 ± 1.37 16.66 ± 2.95 Growth culture medium without EGF and bFGF with LIF (20 ng/ml) 11.12 ± 2.43 18.13 ± 1.99 35. 06 ± 2.63 Data are the mean ± SE of two independent experiments. Each increment was statistically significant at p < 0.005 (Student t-test); n = 4. DIV = days in vitro; LIF = leukemia inhibitory factor; EGF = epidermal growth factor; bFGF = basic fibroblast growth factor. Open in new tab Discussion Pharmacological or neurosurgical therapies are currently used to treat neurological damages in various neurodegenerative disorders (i.e. Parkinson's disease, Alzheimer's disease, Huntington's disease, etc.), but all these strategies are not efficient in preventing or reverting these progressive neurodegenerative processes. Recently, a new approach has been introduced—the cell therapy [20]. This approach is based on the transplantation of appropriate cells, which must not only be well characterized and biologically and immunologically safe, but also sufficiently numerous to ensure adequate post-transplantation survival, tissue regeneration, and an acceptable degree of functional recovery and/or symptomatic improvement. For the first time we have successfully isolated neural stem cells from the olfactory bulb of adult human subjects. The isolation and characterization of neural stem cells from the human olfactory bulb open up a further interesting therapeutic perspective. The high regenerative potential of this area suggests that the olfactory bulb is an ideal autologous source for neurodegenerative disease. Under our optimized conditions, the stem cells obtained from the olfactory bulb, like embryonic stem cells, proliferate and are capable of differentiating into the three classical neural phenotypes. We suggest that stem cells deriving from this area can be simply explanted by means of partial bulbectomy in patients (with few damaging effects such as anosmia). As a result of our many years of experience in manipulating neural stem cells, we are able to expand these cells considerably and, by means of the addition of LIF, ensure their differentiation into neurons [16], the elective biological source for autotransplantation in various neurodegenerative disorders. In particular, the discovery of a large number of immunoreactive tyrosine hydroxylase structures in the olfactory bulbs and peduncles of elderly humans [21] suggests that the olfactory bulb is a hypothetical source for the autotransplantation therapy in Parkinson's disease. In support of the idea of using olfactory bulb neural stem cells for autologous transplantation in patients with Parkinson's disease, we have recently used an experimental model of Parkinson's disease. Lesions in the nigrostriatal zone were induced by inoculation of 6-hidroxy-dopamine in CD1 mice, and we investigated the ability of these cells to elicit functional recovery after intrastriatal transplantation. These data [22, 23] show that inoculation of these cells induces functional recovery in comparison with control untransplanted mice, and detailed biochemical and immunohistochemical evaluations are currently under way. These are fundamental confirmatory data for the future use of these cells for transplant therapy in patients with Parkinson's disease and other neurodegenerative disorders. Conclusion The importance of this discovery is the possibility of obtaining neural stem cells from adult human subjects. It also opens up a new and innovative prospective for the hypothesis of autotransplantation for neurodegenerative diseases. 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TI - Isolation and Characterization of Neural Stem Cells from the Adult Human Olfactory Bulb
JF - Stem Cells
DO - 10.1634/stemcells.18-4-295
DA - 2000-07-01
UR - https://www.deepdyve.com/lp/oxford-university-press/isolation-and-characterization-of-neural-stem-cells-from-the-adult-MSUpnFoX13
SP - 295
EP - 300
VL - 18
IS - 4
DP - DeepDyve
ER -