- Email: [email protected]
Isolation of human adult olfactory sphere cells as a cell source of neural progenitors
Stem Cell Research (2015) 15, 23–29
Available online at www.sciencedirect.com
ScienceDirect www.elsevier.com/locate/scr
METHODS AND REAGENTS
Isolation of human adult olfactory sphere cells as a cell source of neural progenitors☆ Yu-ichiro Ohnishi a,⁎, Koichi Iwatsuki a , Masahiro Ishihara a , Takashi Shikina b , Koei Shinzawa c , Takashi Moriwaki a , Koshi Ninomiya a , Toshika Ohkawa a , Masao Umegaki a , Haruhiko Kishima a , Toshiki Yoshimine a a
Department of Neurosurgery, Osaka University Medical School, 2–2 Yamadaoka, Suita, Osaka 565–0871, Japan Department of Otorhinolaryngology, Osaka University Medical School, 2–2 Yamadaoka, Suita, Osaka 565–0871, Japan c Department of Molecular Genetics, Osaka University Medical School, 2–2 Yamadaoka, Suita, Osaka 565–0871, Japan b
Received 3 March 2015; received in revised form 14 April 2015; accepted 22 April 2015 Available online 29 April 2015
Abstract Olfactory stem cells are generated from olfactory mucosa. Various culture conditions generate olfactory stem cells that differ according to species and developmental stage and have different progenitor or stem cell characteristics. Olfactory spheres (OSs) are clusters of progenitor or stem cells generated from olfactory mucosa in suspension culture. In this study, adult human OSs were generated and their characteristics analyzed. Human OSs were adequately produced from olfactory mucosa with area over 40 mm2. Immunocytochemistry (ICC) and fluorescence-activated cell sorting showed that human OSs were AN2 and A2B5-positive. Immunofluorescence analysis of cell type-specific ICC indicated that the number of Tuj1-positive OS cells was significantly elevated. Tuj1-positive cells displayed typical neuronal soma and dendritic morphology. Human OS cells were also immunopositive for MAP2. By contrast, few RIP-, O4-, and GFAP-positive cells were present. These RIP, O4, and GFAP-positive cells did not resemble bona fide oligodendrocytes and astrocytes morphologically. In culture to induce differentiation of oligodendrocytes, human OS cells also expressed neuronal markers, but neither oligodendrocyte or astrocyte markers. These findings suggest that human OS cells autonomously differentiate into neurons in our culture condition and have potential to be used as a cell source of neural progenitors for their own regenerative grafts, avoiding the need for immunosuppression and ethical controversies. © 2015 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Abbreviations: A2B5, A2B5 antigen; AN2, AN2 proteoglycan; CNS, central nervous system; DAPI, 4′,6-diamidino-2-phenylindole dihydrochloride; DMEM, Dulbecco's modification of Eagle's medium; EDTA, ethylenediaminetetraacetic acid; FACS, fluorescence-activated cell sorting; FBS, fetal bovine serum; FITC, fluorescein isothiocyanate; GAD, glutamic acid decarboxylase; GFAP, glial fibrillary acidic protein; ICC, immunocytochemistry; MAP2, microtubule associated protein 2; NF, neurofilament; Olig2, oligodendrocyte transcription factor 2; OPC, oligodendrocyte progenitor cell; OSCs, olfactory sphere cells; PFA, paraformaldehyde; TH, tyrosine hydroxylase; Tuj1, neuron specific class III beta-tubulin; RIP, receptor interacting protein. ☆ Funding: This study was supported by a Grant-in-Aid for Scientists (C) (25462215). ⁎ Corresponding author. Fax: + 81 6 0879 3659. E-mail address: [email protected] (Y. Ohnishi). http://dx.doi.org/10.1016/j.scr.2015.04.006 1873-5061/© 2015 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
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Introduction The olfactory neurons renew itself throughout life and have potential application in central nervous system (CNS) repair (Barnett and Riddell, 2004; Li et al., 1997; Roisen et al., 2001). Olfactory mucosa is easily accessible, and can be obtained by a simple biopsy performed through the external nares (Feron et al., 1998). Olfactory stem cells are generated from olfactory mucosa (Murdoch and Roskams, 2008; Murrell et al., 2005; Tome et al., 2009). Various culture conditions generate olfactory stem cells that differ according to species and developmental stage and have different progenitor or stem cell characteristics (Lindsay et al., 2013; Murdoch and Roskams, 2008; Murrell et al., 2005; Tome et al., 2009; Zhang et al., 2004). Murrell et al. reported that cells from human olfactory mucosa generate neurospheres that are multipotent in vitro and human olfactory neurospheres gave rise to neurons and glia and self replicating (Murrell et al., 2005, 2008). Olfactory spheres (OSs) are clusters of progenitors or stem cells generated from olfactory mucosa in suspension culture. We previously reported that adult rat olfactory sphere cells (OSCs) were generated, expressed oligodendrocyte progenitor cell (OPC) markers, and differentiated into oligodendrocytes in vivo and in vitro (Ohnishi et al., 2013). Under standard culture conditions, adult rat OSCs rarely differentiate into neurons. In the present study, human adult OSCs were generated and their characteristics investigated. Human adult OSCs also expressed OPC markers, but they were directed toward neuronal differentiation in our culture condition. These findings suggest that human OSCs have potential to be used as a cell source of neural progenitors.
Materials and methods Experimental design This study was approved by the Ethics Committee of the Osaka University Medical School in Osaka, Japan. All procedures were performed after obtaining written, informed consent, which included permission to culture and analyze a biopsy from the tissue to be grafted. Human olfactory mucosa was obtained from surplus mucosa, which was not used for transplantation surgery into spinal cord lesions (Iwatsuki et al., 2013; Lima et al., 2010, Lima et al., 2006).
Collection of olfactory mucosa in humans The otolaryngologists harvested the olfactory mucosa. A transnasal endoscopic approach and instrumentation were used. After cleaning the nasal and olfactory space, vasoconstrictors were injected into the mucosa. A submucoperiosteal tunnel was created in the medial nasal septal side of the olfactory groove and superior concha. Reabsorbable packing was placed in the olfactory groove to avoid postoperative nasal bleeding.
Olfactory sphere culture The procedure of generating human OSs essentially followed that reported by Ohnishi et al. (Ohnishi et al., 2013). In
Y. Ohnishi et al. brief, the olfactory mucosa was carefully dissociated mechanically and then treated for 60 min at 37°C with an enzyme solution comprised of collagenase (Wako, Osaka, Japan), dispase (Sanko Junyaku, Tokyo, Japan), DNase I (Sigma-Aldrich, St Louis, MO), and hyaluronidase (Sigma-Aldrich) in DMEM/F12 medium (DF; Invitrogen Corporation, Carlsbad, CA). Culture dishes were coated with poly(2-hydroxyethyl methacrylate) (Sigma-Aldrich, P3932) to prevent attachment of cells to the bottom of the dish. Dissociated cells were plated onto poly(2-hydroxyethyl methacrylate)-coated dishes in DF medium containing 10% FBS (Gibco), B27 supplement (Invitrogen), 20 ng/mL basic fibroblast growth factor (Sigma-Aldrich), 20 ng/mL epidermal growth factor (Sigma-Aldrich), and antibiotic-antimycotic solution (Invitrogen, 15240) at approximately 5 × 106 cells per 10-cm dish. Cultures were incubated at 37°C in a 5% CO2 atmosphere, and the medium was changed every 3 to 4 days. Cell clusters were classified as OSs if they were spherical in shape and had a diameter of at least 50 μm (Fig. 1A).
Differentiation culture After 10 to 14 days in culture, OSs were treated with trypsin-EDTA (Invitrogen), plated onto poly-L-lysine/ laminin-coated 4-well chamber slides (Becton Dickinson, Franklin Lakes, NJ) at approximately 1 × 103 cells per well, and cultured for 14 to 21 days in DF medium supplemented with 10% FBS (Gibco), N2 (Invitrogen), B27, and antibiotic-antimycotic solution. To induce differentiation into oligodendrocytes, OSCs were cultured in Sato differentiation medium (DMEM supplemented with 2 mM glutaMAX-I, 1 mM sodium pyruvate, 30 nM 3,5,3′-triiodothyronine, 30 nM thyroxine, 1% N2 supplement, 50 U/mL penicillin and 50 μg/mL streptomycin) for 14 to 21 days.
Immunocytochemistry OSCs were fixed with 4% paraformaldehyde (PFA) and incubated for 1 h at room temperature in blocking solution, incubated overnight at 4°C with the primary antibody, washed, and incubated overnight at 4°C in the appropriate species-directed secondary antibody. The primary antibodies were as follows: anti-AN2 biotin conjugated (1:11; Miltenyi Biotec Inc., Auburn, CA), anti-A2B5 biotin conjugated (1:11; Miltenyi Biotec Inc.), anti-β3-tubulin (Tuj1, 1:200 mouse monoclonal antibody; Abcam, Cambridge, UK or 1:200 rabbit polyclonal antibody; Abcam), anti-glutamic acid decarboxylase 67 (GAD67, 1:500 mouse monoclonal antibody; Millipore), anti-glial fibrillary acidic protein (GFAP, 1:300 mouse monoclonal antibody; Cell Signaling Technology), anti-microtubule-associated protein 2 (MAP2, 1:200 rabbit polyclonal antibody, Abcam), anti-neurofilament-L (NF, 1:100 rabbit monoclonal antibody; Cell Signaling Technology), anti-receptor-interacting protein (RIP, 1:100 rabbit monoclonal antibody; Cell Signaling Technology), anti-oligodendrocyte transcription factor 2 (Olig2, 1:300 sheep polyclonal antibody; Abcam), and anti-O4 (1:200 mouse monoclonal; Neuromics, Edina, MN), anti-tyrosine hydroxylase (TH, 1:100 rabbit polyclonal antibody; Abcam). The secondary antibodies used were anti-biotin FITC conjugated (1:100; Miltenyi Biotec Inc.), DyLight 488-conjugated goat
Isolation of human olfactory sphere cells
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Figure 1 Human adult olfactory spheres express oligodendrocyte progenitor markers. (A) Phase-bright olfactory spheres after 14 days in vitro. Olfactory spheres are immunopositive for AN2 (B), and A2B5 (C), markers for oligodendrocyte progenitors. Histograms of FITC-fluorescence emission show that OS cells are weakly positive for A2B5. Scale bar = 50 μm.
anti-mouse (1:200; Kirkegaard and Perry Laboratories, Inc. (KPL), Gaithersburg, MD), DyLight 549-conjugated goat anti-rabbit (1:200; KPL), and Cy5-conjugated donkey anti-sheep (1:200; Jackson ImmunoResearch, West Grove, PA). To visualize cell nuclei, slides were then counterstained with 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI, Vector Laboratories, Burlingame, CA). Cell phenotype assessments and cell counts were obtained from images captured using a confocal laser fluorescence microscope (FV-1000D; Olympus, Tokyo, Japan). The percentages of Tuj1-, RIP-, O4-, NF-, and GFAP-positive cells are shown as means ± standard deviation (SD) of three images from three independent experiments. Primary and secondary antibodies are listed in Table 1.
Fluorescence-activated cell sorting analyses (FACS) Fluorescence-activated cell sorting was performed on a FACS Aria II (BD Biosciences, San Jose, CA). Antibodies were as follows: anti-A2B5-biotin (Miltenyi Biotec), anti-mouse IgM-biotin (Miltenyi Biotec), and anti-biotin-FITC (Miltenyi Biotec). OSs were dissociated with papain for 10 min at 37°C. OS cells (1 × 105) were first reacted with antibodies against A2B5 for 60 min at 4°C and washed. Cells were further
incubated with anti-biotin FITC antibody for 30 min at 4°C. Cells were washed, resuspended, and then passed through a 40-μm filter immediately before sorting. Data were
Table 1 Primary antibodies
List of antibodies used. Target
A2B5-biotin Oligodendrocyte Progenitor cells AN2-biotin Oligodendrocyte progenitor cells GAD67 Neurons GFAP Astrocytes IgM-biotin Control MAP2 Neuron NF Neurons O4 Oligodendrocytes Olig2 Oligodendrocyte/ astrocytes RIP Oligodendrocytes TH Neurons Tuj1 Neurons
Host
Code/ clone
Rat
105-HB29 1/100
Rat
1E6.4
1/100
Mouse Mouse Mouse Rabbit Rabbit Mouse Sheep
MAB5406 ab10062 IS5-20C4 ab32454 C28E10 MO15002 ab85900
1/500 1/250 1/100 1/200 1/100 1/200 1/200
Rabbit D94C12 Rabbit ab122 Mouse ab14545
Dilution
1/100 1/100 1/200
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analyzed using FLOWJO software v6.2.1 (Tree Star, Ashland, OR).
Statistical analyses Data were analyzed by one-way analysis of variance (ANOVA). In the case of a significant F ratio, Tukey's post hoc test was performed. The level of significance was set at P b 0.05, and data are presented as means ± standard deviation unless otherwise indicated. Data were obtained from at least three independent experiments.
Results Harvesting human olfactory mucosa Human olfactory mucosa was obtained from surplus mucosa that was not used for transplantation surgery into spinal cord lesions (Iwatsuki et al., 2013). All surplus mucosa was obtained with the informed consent of the patients, and the study was carried out under a protocol that was approved by the ethics committees of Osaka University Medical School in Osaka Japan. The area of obtained mucosa, and details of each donor are described in Table 2. Five patients (4 males and 1 female, aged 20–40 years) underwent surgery for olfactory mucosal transplantation. The area of surplus olfactory mucosa in 2 patients (patients 1 and 4) and 3 patients (patients 2, 3 and 5) was under 15 mm2 and over 40 mm2, respectively. The yield of primary olfactory spheres was 0 and 2 in patients 1 and 4, respectively. The yield of primary olfactory spheres was over 50 in patients 2, 3, and 5, respectively. The location of surplus olfactory mucosa was the nasal septum in 4 patients and the superior concha in 1 patient.
Human adult OSs express oligodendrocyte progenitor cell markers To determine whether human OSs also expressed oligodendrocyte progenitor markers, immunocytochemistry and FACS using antibodies specific for A2B5 and AN2 were performed. Human OSs were AN2 and A2B5-positive, as measured by immunofluorescence staining (Figs. 1B,C). As further confirmation of these results, FACS analysis revealed the A2B5 proteins were also positive (Fig. 1D). These data indicated that human adult OSs express OPC markers, the same as adult rat OSCs.
Table 2
Patient Patient Patient Patient Patient
Human adult OSCs differentiated into neurons, but neither oligodendrocytes nor astrocytes The differentiation of human adult OSCs was next investigated by immunocytochemistry with antibodies against neuronal and oligoglial markers. Interestingly, immunofluorescence analysis of cell type-specific ICC indicated that the number of Tuj1-positive neuronal cells in human OSCs was significantly elevated (Fig. 2E). Human OSCs were also immunopositive for MAP2 (Fig. 2F), but weak expression than Tuj1. None of GAD67-, and TH-positive cells were present (data not shown). The percentage of Tuj1-positive cells was 87.2 ± 11.7 (mean ± SD) (Fig. 3). Tuj1-positive cells displayed typical neuronal soma and dendritic morphology (Fig. 2E). By contrast, few RIP-, O4-, Olig2, and GFAP-positive cells were present (Figs. 2A–D). The percentages of O4-, and GFAP-positive cells were 4.2% ± 4.4% and 1.5% ± 2.5% (mean ± SD), respectively (Fig. 3). These RIP, O4, and GFAP-positive cells did not resemble bona fide oligodendrocytes and astrocytes morphologically. To test the capacity of differentiation into oligodendrocytes, human OSCs were cultured in Sato differentiation medium. The expressions of NF, O4, and GFAP in OSCs were also investigated by ICC. Human OSCs expressed neuronal markers, but neither oligodendrocyte nor astrocyte markers (Figs. 4A–C). The percentages of NF-, O4-, and GFAP-positive cells were 84.6% ± 16.1%, 6.3% ± 2.2%, and 0% (mean ± SD), respectively (Fig. 4D). Taken together, these findings indicated that human adult OSCs predominantly differentiated into immature or mature neurons.
Discussion Human olfactory stem cells can be grown in neurospheres (Reynolds and Weiss, 1992). Murrell et al. obtained human mucosa by biopsy and generated OSs. They separated olfactory mucosa into lamina propria and epithelium. After separately dissociated, cells of both tissues resuspended into the same culture dish pretreated poly-L-lysine. Their human OSs expressed nestin, GFAP, and Tuj1, and gave rise to neuron and glia under various culture conditions to induce differentiation (Murrell et al., 2005, 2008). When their OSs were dissociated and plated at low density, differentiated cells were observed to express GFAP, O4, Tuj1, and MAP5. The majority of their OSs (50.3% ± 8.09%) expressed GFAP in culture of serum free medium. Fetal calf serum decreased the proportion of cells expressing GFAP (44.67% ± 1.76%) and increased the proportions of cells expressing Tuj1
Mucosal area and location and the yield of primary olfactory spheres.
1 2 3 4 5
Age (y)/sex
Area of donor surplus olfactory mucosa (mm2)
Number of primary olfactory spheres/dish
Location of harvesting olfactory mucosa
28/male 37/female 34/male 30/male 20/male
10 40 50 15 100
0 N 50 N 50 1–2 N 50
Nasal septum Nasal septum Nasal septum Superior concha Nasal septum
Isolation of human olfactory sphere cells
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Figure 2 In vitro differentiation of human OSCs. OSCs are immunopositive for Tuj1 (E) and MAP2 (F). Few are immunopositive for O4 (A), RIP (B), Olig2 (C), and GFAP (D). Scale bar = 50 μm.
(18.33% ± 0.88%) and O4 (3.67% ± 0.88%). CNTF, NGF and retinoic acid increased the proportion of cells expressing GFAP (69.00% ± 3.21%), Tuj1 (25.33% ± 1.45%), and O4 (50.67% ± 2.96%), respectively. They also presented that their human OSCs differentiated into non-neural lineages in vitro and in vivo when given the appropriate signals and
Figure 3 Quantification of O4, GFAP, and Tuj1-positive human OS cells under normal standard differentiation culture. The percentage represents the mean ± SD of 3 independent experiments. *P b 0.05, error bars indicate SD.
environments. In this study, we dissociated olfactory mucosa as a whole, and cells of dissociated tissues resuspended in the culture dish pretreated poly-HEMA. The percentage of Tuj1-positive cells was 87.2% ± 11.7%. These suggest that our human OSs more autonomously differentiate into neurons in our culture condition. Delorme et al. reported that the subtypes of their OSs had the character of mesenchymal stem cells that could help preserve auditory function (Delorme et al., 2010; Feron et al., 2013). These mesenchymal subtypes of their OSs were transplanted into mouse hippocampus, and then they could also induce neurogenesis (Nivet et al., 2011). Mesenchymal OSCs support the survival and differentiation of human hematopoietic stem cells (Diaz-Solano et al., 2012). The lamina propria derived-OSCs have MSC-like properties and secrete factors that enhance both OPC and OEC proliferation (Lindsay et al., 2013). Roisen et al. reported that human olfactory stem cells were harvested from cadavers and patients (Roisen et al., 2001; Winstead et al., 2005). They produced OSs, which contained two subpopulations based on neuronal and glial markers (Roisen et al., 2001). Their OSs consisted of heterogeneous populations in which some cells expressed nestin, A2B5, Tuj1, and peripherin (Zhang et al., 2004). A subset of their A2B5-positive OSCs co-expressed nestin and Tuj1. They also demonstrated that adult human OSCs could be directed toward neuronal lineage restriction by retinoic acid, forskolin, and sonic hedgehog (Zhang et al., 2006).
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Figure 4 Human OSCs were cultured in Sato differentiation medium to induce oligodendrocyte differentiation. (A–C) OSCs express neuronal markers, but neither oligodendrocyte nor astrocyte markers. (D) The percentage represents the mean ± SD of 3 independent experiments. *P b 0.05, Scale bar = 50 μm. Error bars indicate SD.
The probability of finding olfactory epithelium in a biopsy specimen is from 30% to 76%, depending on its location (Feron et al., 1998). In this study, the location of surplus olfactory mucosa was the nasal septum in 4 patients and the superior concha in 1 patient. Murrell et al. produced 500 to 2000 spheres from 1 to 4 mm2 olfactory mucosa after 7– 10 days culture. The number of primary OSs ranged from 0 to 2/culture dish in a harvesting area under 15 mm2, while the number of primary OSs was over 50/culture dish in a harvesting area over 40 mm2. Although generating adequate OSCs seems to require an area of olfactory mucosa over 4 mm × 10 mm, our methods need the improvement of the yield, and scaling down of culture.
Conclusions In this study, adult human OSCs were generated and their characteristics analyzed. Human OSCs were adequately produced from olfactory mucosa with area over 40 mm2. Human OSCs autonomously differentiated into neurons. These findings suggest that human OSCs have the potential to be used as a cell source of neural progenitors for their own regenerative grafts, avoiding the need for immunosuppression and ethical controversies.
Acknowledgments The authors would like to thank Y. Takahashi for administrative assistance. The authors indicate no potential conflicts of interest.
References Barnett, S.C., Riddell, J.S., 2004. Olfactory ensheathing cells (OECs) and the treatment of CNS injury: advantages and possible caveats. J. Anat. 204 (1), 57–67. Delorme, B., Nivet, E., Gaillard, J., Haupl, T., Ringe, J., Deveze, A., Magnan, J., Sohier, J., Khrestchatisky, M., Roman, F.S., Charbord, P., Sensebe, L., Layrolle, P., Feron, F., 2010. The human nose harbors a niche of olfactory ectomesenchymal stem cells displaying neurogenic and osteogenic properties. Stem Cells Dev. 19 (6), 853–866. Diaz-Solano, D., Wittig, O., Ayala-Grosso, C., Pieruzzini, R., Cardier, J.E., 2012. Human olfactory mucosa multipotent mesenchymal stromal cells promote survival, proliferation, and differentiation of human hematopoietic cells. Stem Cells Dev. 21 (17), 3187–3196. Feron, F., Perry, C., McGrath, J.J., Mackay-Sim, A., 1998. New techniques for biopsy and culture of human olfactory epithelial neurons. Arch. Otolaryngol. Head Neck Surg. 124 (8), 861–866. Feron, F., Perry, C., Girard, S.D., Mackay-Sim, A., 2013. Isolation of adult stem cells from the human olfactory mucosa. Methods Mol. Biol. 1059, 107–114.
Isolation of human olfactory sphere cells Iwatsuki, K., Yoshimine, T., Sankai, Y., Tajima, F., Umegaki, M., Ohnishi, Y., Ishihara, M., Ninomiya, K., Moriwaki, T., 2013. Involuntary muscle spasm expressed as motor evoked potential after olfactory mucosa autograft in patients with chronic spinal cord injury and complete paraplegia. J. Biomed. Sci. Eng. 6, 908–916. Li, Y., Field, P.M., Raisman, G., 1997. Repair of adult rat corticospinal tract by transplants of olfactory ensheathing cells. Science 277 (5334), 2000–2002. Lima, C., Pratas-Vital, J., Escada, P., Hasse-Ferreira, A., Capucho, C., Peduzzi, J.D., 2006. Olfactory mucosa autografts in human spinal cord injury: a pilot clinical study. J. Spinal Cord Med. 29 (3), 191–203 (discussion 204-196). Lima, C., Escada, P., Pratas-Vital, J., Branco, C., Arcangeli, C.A., Lazzeri, G., Maia, C.A., Capucho, C., Hasse-Ferreira, A., Peduzzi, J.D., 2010. Olfactory mucosal autografts and rehabilitation for chronic traumatic spinal cord injury. Neurorehabil. Neural Repair 24 (1), 10–22. Lindsay, S.L., Johnstone, S.A., Mountford, J.C., Sheikh, S., Allan, D.B., Clark, L., Barnett, S.C., 2013. Human mesenchymal stem cells isolated from olfactory biopsies but not bone enhance CNS myelination in vitro. Glia 61 (3), 368–382. Murdoch, B., Roskams, A.J., 2008. A novel embryonic nestin-expressing radial glia-like progenitor gives rise to zonally restricted olfactory and vomeronasal neurons. J. Neurosci. 28 (16), 4271–4282. Murrell, W., Feron, F., Wetzig, A., Cameron, N., Splatt, K., Bellette, B., Bianco, J., Perry, C., Lee, G., Mackay-Sim, A., 2005. Multipotent stem cells from adult olfactory mucosa. Dev. Dyn. 233 (2), 496–515. Murrell, W., Wetzig, A., Donnellan, M., Feron, F., Burne, T., Meedeniya, A., Kesby, J., Bianco, J., Perry, C., Silburn, P.,
29 Mackay-Sim, A., 2008. Olfactory mucosa is a potential source for autologous stem cell therapy for Parkinson's disease. Stem Cells 26 (8), 2183–2192. Nivet, E., Vignes, M., Girard, S.D., Pierrisnard, C., Baril, N., Deveze, A., Magnan, J., Lante, F., Khrestchatisky, M., Feron, F., Roman, F.S., 2011. Engraftment of human nasal olfactory stem cells restores neuroplasticity in mice with hippocampal lesions. J. Clin. Invest. 121 (7), 2808–2820. Ohnishi, Y., Iwatsuki, K., Shinzawa, K., Ishihara, M., Moriwaki, T., Umegaki, M., Kishima, H., Yoshimine, T., 2013. Adult olfactory sphere cells are a source of oligodendrocyte and Schwann cell progenitors. Stem Cell Res. 11 (3), 1178–1190. Reynolds, B.A., Weiss, S., 1992. Generation of neurons and astrocytes from isolated cells of the adult mammalian central nervous system. Science 255 (5052), 1707–1710. Roisen, F.J., Klueber, K.M., Lu, C.L., Hatcher, L.M., Dozier, A., Shields, C.B., Maguire, S., 2001. Adult human olfactory stem cells. Brain Res. 890 (1), 11–22. Tome, M., Lindsay, S.L., Riddell, J.S., Barnett, S.C., 2009. Identification of nonepithelial multipotent cells in the embryonic olfactory mucosa. Stem Cells 27 (9), 2196–2208. Winstead, W., Marshall, C.T., Lu, C.L., Klueber, K.M., Roisen, F.J., 2005. Endoscopic biopsy of human olfactory epithelium as a source of progenitor cells. Am. J. Rhinol. 19 (1), 83–90. Zhang, X., Klueber, K.M., Guo, Z., Lu, C., Roisen, F.J., 2004. Adult human olfactory neural progenitors cultured in defined medium. Exp. Neurol. 186 (2), 112–123. Zhang, X., Klueber, K.M., Guo, Z., Cai, J., Lu, C., Winstead, W.I., Qiu, M., Roisen, F.J., 2006. Induction of neuronal differentiation of adult human olfactory neuroepithelial-derived progenitors. Brain Res. 1073–1074, 109–119.
Available online at www.sciencedirect.com
ScienceDirect www.elsevier.com/locate/scr
METHODS AND REAGENTS
Isolation of human adult olfactory sphere cells as a cell source of neural progenitors☆ Yu-ichiro Ohnishi a,⁎, Koichi Iwatsuki a , Masahiro Ishihara a , Takashi Shikina b , Koei Shinzawa c , Takashi Moriwaki a , Koshi Ninomiya a , Toshika Ohkawa a , Masao Umegaki a , Haruhiko Kishima a , Toshiki Yoshimine a a
Department of Neurosurgery, Osaka University Medical School, 2–2 Yamadaoka, Suita, Osaka 565–0871, Japan Department of Otorhinolaryngology, Osaka University Medical School, 2–2 Yamadaoka, Suita, Osaka 565–0871, Japan c Department of Molecular Genetics, Osaka University Medical School, 2–2 Yamadaoka, Suita, Osaka 565–0871, Japan b
Received 3 March 2015; received in revised form 14 April 2015; accepted 22 April 2015 Available online 29 April 2015
Abstract Olfactory stem cells are generated from olfactory mucosa. Various culture conditions generate olfactory stem cells that differ according to species and developmental stage and have different progenitor or stem cell characteristics. Olfactory spheres (OSs) are clusters of progenitor or stem cells generated from olfactory mucosa in suspension culture. In this study, adult human OSs were generated and their characteristics analyzed. Human OSs were adequately produced from olfactory mucosa with area over 40 mm2. Immunocytochemistry (ICC) and fluorescence-activated cell sorting showed that human OSs were AN2 and A2B5-positive. Immunofluorescence analysis of cell type-specific ICC indicated that the number of Tuj1-positive OS cells was significantly elevated. Tuj1-positive cells displayed typical neuronal soma and dendritic morphology. Human OS cells were also immunopositive for MAP2. By contrast, few RIP-, O4-, and GFAP-positive cells were present. These RIP, O4, and GFAP-positive cells did not resemble bona fide oligodendrocytes and astrocytes morphologically. In culture to induce differentiation of oligodendrocytes, human OS cells also expressed neuronal markers, but neither oligodendrocyte or astrocyte markers. These findings suggest that human OS cells autonomously differentiate into neurons in our culture condition and have potential to be used as a cell source of neural progenitors for their own regenerative grafts, avoiding the need for immunosuppression and ethical controversies. © 2015 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Abbreviations: A2B5, A2B5 antigen; AN2, AN2 proteoglycan; CNS, central nervous system; DAPI, 4′,6-diamidino-2-phenylindole dihydrochloride; DMEM, Dulbecco's modification of Eagle's medium; EDTA, ethylenediaminetetraacetic acid; FACS, fluorescence-activated cell sorting; FBS, fetal bovine serum; FITC, fluorescein isothiocyanate; GAD, glutamic acid decarboxylase; GFAP, glial fibrillary acidic protein; ICC, immunocytochemistry; MAP2, microtubule associated protein 2; NF, neurofilament; Olig2, oligodendrocyte transcription factor 2; OPC, oligodendrocyte progenitor cell; OSCs, olfactory sphere cells; PFA, paraformaldehyde; TH, tyrosine hydroxylase; Tuj1, neuron specific class III beta-tubulin; RIP, receptor interacting protein. ☆ Funding: This study was supported by a Grant-in-Aid for Scientists (C) (25462215). ⁎ Corresponding author. Fax: + 81 6 0879 3659. E-mail address: [email protected] (Y. Ohnishi). http://dx.doi.org/10.1016/j.scr.2015.04.006 1873-5061/© 2015 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
24
Introduction The olfactory neurons renew itself throughout life and have potential application in central nervous system (CNS) repair (Barnett and Riddell, 2004; Li et al., 1997; Roisen et al., 2001). Olfactory mucosa is easily accessible, and can be obtained by a simple biopsy performed through the external nares (Feron et al., 1998). Olfactory stem cells are generated from olfactory mucosa (Murdoch and Roskams, 2008; Murrell et al., 2005; Tome et al., 2009). Various culture conditions generate olfactory stem cells that differ according to species and developmental stage and have different progenitor or stem cell characteristics (Lindsay et al., 2013; Murdoch and Roskams, 2008; Murrell et al., 2005; Tome et al., 2009; Zhang et al., 2004). Murrell et al. reported that cells from human olfactory mucosa generate neurospheres that are multipotent in vitro and human olfactory neurospheres gave rise to neurons and glia and self replicating (Murrell et al., 2005, 2008). Olfactory spheres (OSs) are clusters of progenitors or stem cells generated from olfactory mucosa in suspension culture. We previously reported that adult rat olfactory sphere cells (OSCs) were generated, expressed oligodendrocyte progenitor cell (OPC) markers, and differentiated into oligodendrocytes in vivo and in vitro (Ohnishi et al., 2013). Under standard culture conditions, adult rat OSCs rarely differentiate into neurons. In the present study, human adult OSCs were generated and their characteristics investigated. Human adult OSCs also expressed OPC markers, but they were directed toward neuronal differentiation in our culture condition. These findings suggest that human OSCs have potential to be used as a cell source of neural progenitors.
Materials and methods Experimental design This study was approved by the Ethics Committee of the Osaka University Medical School in Osaka, Japan. All procedures were performed after obtaining written, informed consent, which included permission to culture and analyze a biopsy from the tissue to be grafted. Human olfactory mucosa was obtained from surplus mucosa, which was not used for transplantation surgery into spinal cord lesions (Iwatsuki et al., 2013; Lima et al., 2010, Lima et al., 2006).
Collection of olfactory mucosa in humans The otolaryngologists harvested the olfactory mucosa. A transnasal endoscopic approach and instrumentation were used. After cleaning the nasal and olfactory space, vasoconstrictors were injected into the mucosa. A submucoperiosteal tunnel was created in the medial nasal septal side of the olfactory groove and superior concha. Reabsorbable packing was placed in the olfactory groove to avoid postoperative nasal bleeding.
Olfactory sphere culture The procedure of generating human OSs essentially followed that reported by Ohnishi et al. (Ohnishi et al., 2013). In
Y. Ohnishi et al. brief, the olfactory mucosa was carefully dissociated mechanically and then treated for 60 min at 37°C with an enzyme solution comprised of collagenase (Wako, Osaka, Japan), dispase (Sanko Junyaku, Tokyo, Japan), DNase I (Sigma-Aldrich, St Louis, MO), and hyaluronidase (Sigma-Aldrich) in DMEM/F12 medium (DF; Invitrogen Corporation, Carlsbad, CA). Culture dishes were coated with poly(2-hydroxyethyl methacrylate) (Sigma-Aldrich, P3932) to prevent attachment of cells to the bottom of the dish. Dissociated cells were plated onto poly(2-hydroxyethyl methacrylate)-coated dishes in DF medium containing 10% FBS (Gibco), B27 supplement (Invitrogen), 20 ng/mL basic fibroblast growth factor (Sigma-Aldrich), 20 ng/mL epidermal growth factor (Sigma-Aldrich), and antibiotic-antimycotic solution (Invitrogen, 15240) at approximately 5 × 106 cells per 10-cm dish. Cultures were incubated at 37°C in a 5% CO2 atmosphere, and the medium was changed every 3 to 4 days. Cell clusters were classified as OSs if they were spherical in shape and had a diameter of at least 50 μm (Fig. 1A).
Differentiation culture After 10 to 14 days in culture, OSs were treated with trypsin-EDTA (Invitrogen), plated onto poly-L-lysine/ laminin-coated 4-well chamber slides (Becton Dickinson, Franklin Lakes, NJ) at approximately 1 × 103 cells per well, and cultured for 14 to 21 days in DF medium supplemented with 10% FBS (Gibco), N2 (Invitrogen), B27, and antibiotic-antimycotic solution. To induce differentiation into oligodendrocytes, OSCs were cultured in Sato differentiation medium (DMEM supplemented with 2 mM glutaMAX-I, 1 mM sodium pyruvate, 30 nM 3,5,3′-triiodothyronine, 30 nM thyroxine, 1% N2 supplement, 50 U/mL penicillin and 50 μg/mL streptomycin) for 14 to 21 days.
Immunocytochemistry OSCs were fixed with 4% paraformaldehyde (PFA) and incubated for 1 h at room temperature in blocking solution, incubated overnight at 4°C with the primary antibody, washed, and incubated overnight at 4°C in the appropriate species-directed secondary antibody. The primary antibodies were as follows: anti-AN2 biotin conjugated (1:11; Miltenyi Biotec Inc., Auburn, CA), anti-A2B5 biotin conjugated (1:11; Miltenyi Biotec Inc.), anti-β3-tubulin (Tuj1, 1:200 mouse monoclonal antibody; Abcam, Cambridge, UK or 1:200 rabbit polyclonal antibody; Abcam), anti-glutamic acid decarboxylase 67 (GAD67, 1:500 mouse monoclonal antibody; Millipore), anti-glial fibrillary acidic protein (GFAP, 1:300 mouse monoclonal antibody; Cell Signaling Technology), anti-microtubule-associated protein 2 (MAP2, 1:200 rabbit polyclonal antibody, Abcam), anti-neurofilament-L (NF, 1:100 rabbit monoclonal antibody; Cell Signaling Technology), anti-receptor-interacting protein (RIP, 1:100 rabbit monoclonal antibody; Cell Signaling Technology), anti-oligodendrocyte transcription factor 2 (Olig2, 1:300 sheep polyclonal antibody; Abcam), and anti-O4 (1:200 mouse monoclonal; Neuromics, Edina, MN), anti-tyrosine hydroxylase (TH, 1:100 rabbit polyclonal antibody; Abcam). The secondary antibodies used were anti-biotin FITC conjugated (1:100; Miltenyi Biotec Inc.), DyLight 488-conjugated goat
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Figure 1 Human adult olfactory spheres express oligodendrocyte progenitor markers. (A) Phase-bright olfactory spheres after 14 days in vitro. Olfactory spheres are immunopositive for AN2 (B), and A2B5 (C), markers for oligodendrocyte progenitors. Histograms of FITC-fluorescence emission show that OS cells are weakly positive for A2B5. Scale bar = 50 μm.
anti-mouse (1:200; Kirkegaard and Perry Laboratories, Inc. (KPL), Gaithersburg, MD), DyLight 549-conjugated goat anti-rabbit (1:200; KPL), and Cy5-conjugated donkey anti-sheep (1:200; Jackson ImmunoResearch, West Grove, PA). To visualize cell nuclei, slides were then counterstained with 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI, Vector Laboratories, Burlingame, CA). Cell phenotype assessments and cell counts were obtained from images captured using a confocal laser fluorescence microscope (FV-1000D; Olympus, Tokyo, Japan). The percentages of Tuj1-, RIP-, O4-, NF-, and GFAP-positive cells are shown as means ± standard deviation (SD) of three images from three independent experiments. Primary and secondary antibodies are listed in Table 1.
Fluorescence-activated cell sorting analyses (FACS) Fluorescence-activated cell sorting was performed on a FACS Aria II (BD Biosciences, San Jose, CA). Antibodies were as follows: anti-A2B5-biotin (Miltenyi Biotec), anti-mouse IgM-biotin (Miltenyi Biotec), and anti-biotin-FITC (Miltenyi Biotec). OSs were dissociated with papain for 10 min at 37°C. OS cells (1 × 105) were first reacted with antibodies against A2B5 for 60 min at 4°C and washed. Cells were further
incubated with anti-biotin FITC antibody for 30 min at 4°C. Cells were washed, resuspended, and then passed through a 40-μm filter immediately before sorting. Data were
Table 1 Primary antibodies
List of antibodies used. Target
A2B5-biotin Oligodendrocyte Progenitor cells AN2-biotin Oligodendrocyte progenitor cells GAD67 Neurons GFAP Astrocytes IgM-biotin Control MAP2 Neuron NF Neurons O4 Oligodendrocytes Olig2 Oligodendrocyte/ astrocytes RIP Oligodendrocytes TH Neurons Tuj1 Neurons
Host
Code/ clone
Rat
105-HB29 1/100
Rat
1E6.4
1/100
Mouse Mouse Mouse Rabbit Rabbit Mouse Sheep
MAB5406 ab10062 IS5-20C4 ab32454 C28E10 MO15002 ab85900
1/500 1/250 1/100 1/200 1/100 1/200 1/200
Rabbit D94C12 Rabbit ab122 Mouse ab14545
Dilution
1/100 1/100 1/200
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analyzed using FLOWJO software v6.2.1 (Tree Star, Ashland, OR).
Statistical analyses Data were analyzed by one-way analysis of variance (ANOVA). In the case of a significant F ratio, Tukey's post hoc test was performed. The level of significance was set at P b 0.05, and data are presented as means ± standard deviation unless otherwise indicated. Data were obtained from at least three independent experiments.
Results Harvesting human olfactory mucosa Human olfactory mucosa was obtained from surplus mucosa that was not used for transplantation surgery into spinal cord lesions (Iwatsuki et al., 2013). All surplus mucosa was obtained with the informed consent of the patients, and the study was carried out under a protocol that was approved by the ethics committees of Osaka University Medical School in Osaka Japan. The area of obtained mucosa, and details of each donor are described in Table 2. Five patients (4 males and 1 female, aged 20–40 years) underwent surgery for olfactory mucosal transplantation. The area of surplus olfactory mucosa in 2 patients (patients 1 and 4) and 3 patients (patients 2, 3 and 5) was under 15 mm2 and over 40 mm2, respectively. The yield of primary olfactory spheres was 0 and 2 in patients 1 and 4, respectively. The yield of primary olfactory spheres was over 50 in patients 2, 3, and 5, respectively. The location of surplus olfactory mucosa was the nasal septum in 4 patients and the superior concha in 1 patient.
Human adult OSs express oligodendrocyte progenitor cell markers To determine whether human OSs also expressed oligodendrocyte progenitor markers, immunocytochemistry and FACS using antibodies specific for A2B5 and AN2 were performed. Human OSs were AN2 and A2B5-positive, as measured by immunofluorescence staining (Figs. 1B,C). As further confirmation of these results, FACS analysis revealed the A2B5 proteins were also positive (Fig. 1D). These data indicated that human adult OSs express OPC markers, the same as adult rat OSCs.
Table 2
Patient Patient Patient Patient Patient
Human adult OSCs differentiated into neurons, but neither oligodendrocytes nor astrocytes The differentiation of human adult OSCs was next investigated by immunocytochemistry with antibodies against neuronal and oligoglial markers. Interestingly, immunofluorescence analysis of cell type-specific ICC indicated that the number of Tuj1-positive neuronal cells in human OSCs was significantly elevated (Fig. 2E). Human OSCs were also immunopositive for MAP2 (Fig. 2F), but weak expression than Tuj1. None of GAD67-, and TH-positive cells were present (data not shown). The percentage of Tuj1-positive cells was 87.2 ± 11.7 (mean ± SD) (Fig. 3). Tuj1-positive cells displayed typical neuronal soma and dendritic morphology (Fig. 2E). By contrast, few RIP-, O4-, Olig2, and GFAP-positive cells were present (Figs. 2A–D). The percentages of O4-, and GFAP-positive cells were 4.2% ± 4.4% and 1.5% ± 2.5% (mean ± SD), respectively (Fig. 3). These RIP, O4, and GFAP-positive cells did not resemble bona fide oligodendrocytes and astrocytes morphologically. To test the capacity of differentiation into oligodendrocytes, human OSCs were cultured in Sato differentiation medium. The expressions of NF, O4, and GFAP in OSCs were also investigated by ICC. Human OSCs expressed neuronal markers, but neither oligodendrocyte nor astrocyte markers (Figs. 4A–C). The percentages of NF-, O4-, and GFAP-positive cells were 84.6% ± 16.1%, 6.3% ± 2.2%, and 0% (mean ± SD), respectively (Fig. 4D). Taken together, these findings indicated that human adult OSCs predominantly differentiated into immature or mature neurons.
Discussion Human olfactory stem cells can be grown in neurospheres (Reynolds and Weiss, 1992). Murrell et al. obtained human mucosa by biopsy and generated OSs. They separated olfactory mucosa into lamina propria and epithelium. After separately dissociated, cells of both tissues resuspended into the same culture dish pretreated poly-L-lysine. Their human OSs expressed nestin, GFAP, and Tuj1, and gave rise to neuron and glia under various culture conditions to induce differentiation (Murrell et al., 2005, 2008). When their OSs were dissociated and plated at low density, differentiated cells were observed to express GFAP, O4, Tuj1, and MAP5. The majority of their OSs (50.3% ± 8.09%) expressed GFAP in culture of serum free medium. Fetal calf serum decreased the proportion of cells expressing GFAP (44.67% ± 1.76%) and increased the proportions of cells expressing Tuj1
Mucosal area and location and the yield of primary olfactory spheres.
1 2 3 4 5
Age (y)/sex
Area of donor surplus olfactory mucosa (mm2)
Number of primary olfactory spheres/dish
Location of harvesting olfactory mucosa
28/male 37/female 34/male 30/male 20/male
10 40 50 15 100
0 N 50 N 50 1–2 N 50
Nasal septum Nasal septum Nasal septum Superior concha Nasal septum
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Figure 2 In vitro differentiation of human OSCs. OSCs are immunopositive for Tuj1 (E) and MAP2 (F). Few are immunopositive for O4 (A), RIP (B), Olig2 (C), and GFAP (D). Scale bar = 50 μm.
(18.33% ± 0.88%) and O4 (3.67% ± 0.88%). CNTF, NGF and retinoic acid increased the proportion of cells expressing GFAP (69.00% ± 3.21%), Tuj1 (25.33% ± 1.45%), and O4 (50.67% ± 2.96%), respectively. They also presented that their human OSCs differentiated into non-neural lineages in vitro and in vivo when given the appropriate signals and
Figure 3 Quantification of O4, GFAP, and Tuj1-positive human OS cells under normal standard differentiation culture. The percentage represents the mean ± SD of 3 independent experiments. *P b 0.05, error bars indicate SD.
environments. In this study, we dissociated olfactory mucosa as a whole, and cells of dissociated tissues resuspended in the culture dish pretreated poly-HEMA. The percentage of Tuj1-positive cells was 87.2% ± 11.7%. These suggest that our human OSs more autonomously differentiate into neurons in our culture condition. Delorme et al. reported that the subtypes of their OSs had the character of mesenchymal stem cells that could help preserve auditory function (Delorme et al., 2010; Feron et al., 2013). These mesenchymal subtypes of their OSs were transplanted into mouse hippocampus, and then they could also induce neurogenesis (Nivet et al., 2011). Mesenchymal OSCs support the survival and differentiation of human hematopoietic stem cells (Diaz-Solano et al., 2012). The lamina propria derived-OSCs have MSC-like properties and secrete factors that enhance both OPC and OEC proliferation (Lindsay et al., 2013). Roisen et al. reported that human olfactory stem cells were harvested from cadavers and patients (Roisen et al., 2001; Winstead et al., 2005). They produced OSs, which contained two subpopulations based on neuronal and glial markers (Roisen et al., 2001). Their OSs consisted of heterogeneous populations in which some cells expressed nestin, A2B5, Tuj1, and peripherin (Zhang et al., 2004). A subset of their A2B5-positive OSCs co-expressed nestin and Tuj1. They also demonstrated that adult human OSCs could be directed toward neuronal lineage restriction by retinoic acid, forskolin, and sonic hedgehog (Zhang et al., 2006).
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Figure 4 Human OSCs were cultured in Sato differentiation medium to induce oligodendrocyte differentiation. (A–C) OSCs express neuronal markers, but neither oligodendrocyte nor astrocyte markers. (D) The percentage represents the mean ± SD of 3 independent experiments. *P b 0.05, Scale bar = 50 μm. Error bars indicate SD.
The probability of finding olfactory epithelium in a biopsy specimen is from 30% to 76%, depending on its location (Feron et al., 1998). In this study, the location of surplus olfactory mucosa was the nasal septum in 4 patients and the superior concha in 1 patient. Murrell et al. produced 500 to 2000 spheres from 1 to 4 mm2 olfactory mucosa after 7– 10 days culture. The number of primary OSs ranged from 0 to 2/culture dish in a harvesting area under 15 mm2, while the number of primary OSs was over 50/culture dish in a harvesting area over 40 mm2. Although generating adequate OSCs seems to require an area of olfactory mucosa over 4 mm × 10 mm, our methods need the improvement of the yield, and scaling down of culture.
Conclusions In this study, adult human OSCs were generated and their characteristics analyzed. Human OSCs were adequately produced from olfactory mucosa with area over 40 mm2. Human OSCs autonomously differentiated into neurons. These findings suggest that human OSCs have the potential to be used as a cell source of neural progenitors for their own regenerative grafts, avoiding the need for immunosuppression and ethical controversies.
Acknowledgments The authors would like to thank Y. Takahashi for administrative assistance. The authors indicate no potential conflicts of interest.
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