The characteristics of human cranial bone marrow mesenchymal stem cells

The characteristics of human cranial bone marrow mesenchymal stem cells

Neuroscience Letters 606 (2015) 161–166 Contents lists available at ScienceDirect Neuroscience Letters journal homepage: www.elsevier.com/locate/neu...

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Neuroscience Letters 606 (2015) 161–166

Contents lists available at ScienceDirect

Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet

Research paper

The characteristics of human cranial bone marrow mesenchymal stem cells Katsuhiro Shinagawa a,∗ , Takafumi Mitsuhara a , Takahito Okazaki a , Masaaki Takeda a , Satoshi Yamaguchi a , Takuro Magaki b , Yunosuke Okura c , Hiroyuki Uwatoko c , Yumi Kawahara d , Louis Yuge d , Kaoru Kurisu a a Department of Neurosurgery, Graduate School of Biomedical and Health Sciences, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima City, Hiroshima 734-8551, Japan b Department of Neurosurgery, Hiroshima Prefectural Hospital, 1-5-54 Ujinakanda, Minami-ku, Hiroshima City, Hiroshima 734-8530, Japan c Bio-Environmental Adaptation Sciences, Graduate School of Biomedical and Health Sciences, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima City, Hiroshima 734-8551, Japan d Space Bio-Laboratories Co., Ltd., 1-9-14-503 Danbara-minami, Minami-ku, Hiroshima City, Hiroshima 732-0814, Japan

h i g h l i g h t s • We established mesenchymal stem cells from human cranial bone marrow. • We confirmed neural crest-associated gene expression of our stem cells. • These cells should have a greater tendency to differentiate into neuronal cells.

a r t i c l e

i n f o

Article history: Received 1 July 2015 Received in revised form 31 August 2015 Accepted 31 August 2015 Available online 3 September 2015 Keywords: Bone marrow-derived mesenchymal stem cell Cranial bone marrow Neural crest Neuronal differentiation Cell therapy

a b s t r a c t Recently, cell-based therapy has attracted attention for treatment of central nervous system (CNS) disorders. Bone marrow-derived mesenchymal stem cells (BMSCs) are considered to have good engraftment potential. Therefore, more efficient and less invasive methods to obtain donor cells are required. Here, we established human BMSCs from cranial bone waste (cBMSCs) obtained following routine neurosurgical procedures. cBMSCs and cells obtained from the iliac crest (iBMSCs, standard BMSCs) showed expression of cell surface markers associated with mesenchymal stem cells and multipotency traits such as differentiation into osteogenic and adipogenic lineages. cBMSCs showed higher expression of the neural crest-associated mRNAs p75, Slug, and Snail than iBMSCs. Neurogenic induced cells from cBMSCs expressed the neural markers nestin, Pax6, neurofilament (NF)-L, and NF-M as seen with RT-PCR, and NF-M protein as seen with western blotting at higher levels than cells from iBMSCs. Immunostaining showed a significantly greater proportion of NF-M-positive cells in the population of induced cBMSCs compared with the population of iBMSCs. Thus, cBMSCs showed a greater tendency to differentiate into neuron-like cells than iBMSCs. © 2015 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Abbreviations: BMSC, bone marrow-derived mesenchymal stem cell; cBMSC, cranial BMSC; CNS, central nervous system; DAPI, 4 ,6-diamidino-2-phenylindole dihydrochloride; DMEM, Dulbecco’s modified Eagle medium; FBS, fetal bovine serum; G3PDH, glyceraldehyde-3-phosphate dehydrogenase; hBMSC, human BMSC; HLA-DR, human leukocyte antigen-DR; iBMSC, iliac BMSC; mAbs, monoclonal antibodies; MSC, mesenchymal stem cell; NF-L, neurofilament light chain; NF-M, neurofilament medium chain; Pax6, paired box 6; PBS, phosphate-buffered saline; RT-PCR, reverse transcription-polymerase chain reaction; SDS, sodium dodecyl sulfate. ∗ Corresponding author. E-mail address: [email protected] (K. Shinagawa). http://dx.doi.org/10.1016/j.neulet.2015.08.056 0304-3940/© 2015 Elsevier Ireland Ltd. All rights reserved.

Recovery after central nervous system (CNS) disorders such as brain stroke, traumatic brain injury, spinal cord injury, and degenerative disease is often restricted and poor because brain and nervous tissue have a limited ability for self-repair after injury [1,2]. After neuronal stem cells were identified in the adult mammalian brain [3], much attention has recently been focused on regenerative cell therapy for CNS disorders. Currently, mesenchymal stem cells (MSCs) are expected to be candidate cells for grafting. Honmou et al. transplanted autologous

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human bone marrow-derived mesenchymal stem cells (hBMSCs) that were expanded in autologous human serum into stroke patients in a clinical study and showed reductions in neurological deficits and the lesion size. They described the feasibility and safety of this method [4]. Human MSCs secrete neurotrophins such as brain-derived neurotrophic factor and glial cell line-derived neurotrophic factor that contribute to anatomical and functional motor recovery in the ischemic brain [5]. Therefore, selection of cells for grafting that have a greater tendency to differentiate into neuron-like or glial-like cells may provide a maximal therapeutic response in CNS regenerative medicine. The bones of the face and part of the cranial vault are derived from the neural crest, and bones of the limbs and vertebrae are derived from the mesodermal germ layer [6]. Most previous studies that attempted to induce neuronal differentiation of hBMSCs used iliac bone marrow-derived cells, which are derived from the mesodermal germ layer. We have focused on hBMSCs derived from cranial bone marrow, which are obtained from bone waste in neurosurgical procedures. Previous reports suggest that neurogenic potential is an innate characteristic of MSCs, particularly those of dental origin that are derived from cranial neural crest cells, and MSCs derived from human teeth have high neurogenic potential [7]. Based on these findings, hBMSCs from cranial bone, which are derived from the neural crest, are likely to have high neurogenic potential and have been proposed to be a valuable source of stem cells for CNS cell therapy. In this study, we isolated hBMSCs from cranial bone marrow (cBMSCs) and investigated their undifferentiated phenotype and differentiation potential into multi-lineage cell types. To investigate the neurogenic potential of cBMSCs, they were induced to differentiate into neural cells. Then, we analyzed the characteristics of differentiated cBMSCs by comparing them with hBMSCs from iliac crest bone marrow (iBMSCs), which are considered standard BMSCs.

CD105, APC-conjugated primary mAbs against CD73, and PEconjugated primary mAbs against CD44 were used as MSC markers, and FITC-conjugated primary mAbs against CD11b, CD34, CD45, and human leukocyte antigen-DR (HLA-DR) (each from BioLegend Inc., San Diego, CA, USA) were used as endothelial/hematopoietic markers. Data acquisition and analyses were performed with BD FACSAria (BD Biosciences, San Jose, CA, USA). 2.4. Multi-lineage cell differentiation BMSCs were differentiated using osteogenic and adipogenic induction conditions. To induce osteogenic or adipogenic differentiation, cells were seeded at 2.0 × 104 cells/10-cm culture dish and maintained in growth medium until confluent. For osteogenic induction, cells were cultured in DMEM-L containing 10% FBS, 2 mM l-glutamine (Sigma–Aldrich Co.), 100 nM dexamethasone (Sigma–Aldrich), 10 mM ␤-glycerophosphate disodium salt hydrate (Sigma–Aldrich), 50 ␮g/ml L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate (Sigma–Aldrich), penicillin (100 U/ml), and streptomycin (100 ␮g/ml) for 7 days. To visualize the mineralized deposits, the cultures were fixed in 95% ethyl alcohol and stained with 1% alizarin red S followed by microscopic examination using a multifunctional microscope (BZ-9000; KEYENCE Co., Osaka, Japan). For adipogenic induction, the AdvanceSTEM Adipogenic Differentiation Kit (Thermo Fisher Scientific HyClone) was used. Cells were cultured in AdvanceSTEM Adipogenic Differentiation Medium (89%) containing AdvanceSTEM Growth Supplement (5%), penicillin (100 U/ml), and streptomycin (100 ␮g/ml) for 3 weeks. Lipid-laden fat cells were washed twice with phosphate-buffered saline (PBS) and stained with Oil red-O for 10 min at room temperature followed by microscopic examination. Growth and differentiation medium was changed every 3–4 days. 2.5. Neural differentiation

2. Materials and methods 2.1. Tissue sampling Cranial bone marrow samples were obtained from frontotemporal cranial bone waste following neurosurgical procedures after informed consent was obtained from the patient, according to the university hospital’s guidelines. Iliac bone marrow was obtained from iliac bone waste following spinal surgery and served as a control. 2.2. Tissue culture The bone marrow samples were seeded in culture dishes containing low-glucose Dulbecco’s modified Eagle medium (DMEM-L; Sigma–Aldrich Co., St. Louis, MO, USA) with 10% fetal bovine serum (FBS) (Thermo Fisher Scientific HyClone, South Logan, UT, USA), penicillin (100 U/ml), and streptomycin (100 ␮g/ml; both from Sigma–Aldrich). The dishes were maintained at 37 ◦ C in 5% CO2 in a humidified chamber. The medium was changed twice a week to eliminate floating bone powder and non-adherent cells, and adherent cells were incubated until 90% confluent. The cells that adhered to the bottom of the culture dish were used as BMSCs [8] and were passaged several times. 2.3. Flow cytometry analysis for specific cell markers To examine specific cell markers of stem cells derived from cranial bone marrow, flow cytometry analysis was performed. Alexa Fluor-conjugated primary monoclonal antibodies (mAbs) against

After at least four passages, the BMSCs were induced to differentiate into neural cells with neurotropic factors. We used modified neural differentiation conditions including neural conditioning medium and neural differentiation medium according to previously reported methods [9]. Specifically, the BMSCs were seeded at a density of 2.0 × 104 cells/10-cm dish and maintained in growth medium until 80% confluent. Then the medium was changed to the neural conditioning medium containing Dulbecco’s modified Eagle’s/F12 (Invitrogen Co., Carlsbad, CA, USA) with 1% FBS (Thermo Fisher Scientific HyClone), basic fibroblast growth factor (100 ng/ml; PeproTech, Rocky Hill, NJ, USA), penicillin (100 U/ml), and streptomycin (100 ␮g/ml; both from Sigma–Aldrich). After incubating in this neural conditioning medium for 3 days, the cells were cultured in neural differentiation medium composed of neural conditioning medium with forskolin (10 ␮M; Sigma–Aldrich) added for 7 days. The differentiation medium was changed every 3 or 4 days. 2.6. Reverse transcription-polymerase chain reaction (RT-PCR) Cultured cells were collected in ISOGEN (Nippon Gene Co., Ltd., Toyama, Japan), and RNA was isolated according to the manufacturer’s protocol. Reverse transcription was performed with ReverTra Ace-␣ (Toyobo Co., Ltd., Osaka, Japan). Using cDNA as the template, PCR was performed with BD Advantage 2 PCR Kits (BD Biosciences Clontech, Palo Alto, CA, USA). We used nestin as a neural progenitor cell marker, paired box 6 (Pax6) as a neural precursor cell marker, and neurofilament-L (NF-L) and neurofilament-M (NF-M) as neuronal markers. Glyceraldehyde-3-phosphate dehydrogenase

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Table 1 Primer sequences and conditions for amplification of marker genes. Gene name

Primer sequences

PCR conditions

Size (bp)

p75

F: 5 -GTGGGACAGAGTCTGGGTGT-3 R: 5 -AAGGAGGGGAGGTGATAGGA-3 F: 5 -TCGGACCCACACATTACC-3 R: 5 -TCTCTCAATCTAGCCATCAGC-3 F: 5 -TATGCTGCCTTCCCAGGCTTG-3 R: 5 -ATGTGCATCTTGAGGGCACCC-3 F: 5 -GCGTTGGAACAGAGGTTGGAG-3 R: 5 -GCACAGGTGTCTCAAGGGTAG-3 F: 5 -GGTCTGTACCAACGATAACATAC-3 R: 5 -CTGATAGGAATATGACTAGGTGTG-3 F: 5 -TCCTACTACACCAGCCATGT-3 R: 5 -TCCCCAGCACCTTCAACTTT-3 F: 5 -TGGGAAATGGCTCGTCATTT-3 R: 5 -CTTCATGGAAACGGCCAATT-3 F: 5 -TGAAGGTCGGAGTCAACGGATTT-3 R: 5 -CATGTGGGCCATGAGGTCCACCAC-3

95 ◦ C–30 s, 60 ◦ C–30 s, 68 ◦ C–30 s 39 cycles 95 ◦ C–30 s, 59 ◦ C–30 s, 68 ◦ C–45 s 30 cycles 95 ◦ C–30 s, 66 ◦ C–30 s, 68 ◦ C–30 s 27 cycles 95 ◦ C–30 s, 62 ◦ C–30 s, 68 ◦ C–60 s 35 cycles 95 ◦ C–30 s, 62 ◦ C–30 s, 68 ◦ C–60 s 35 cycles 95 ◦ C–30 s, 62 ◦ C–30 s, 68 ◦ C–60 s 35 cycles 95 ◦ C–30 s, 62 ◦ C–30 s, 68 ◦ C–60 s 35 cycles 95 ◦ C–30 s, 55 ◦ C–30 s, 68 ◦ C–60 s 20 cycles

203

Slug Snail Nestin Pax6 NF-L NF-M Glyceraldehyde-3-phosphate dehydrogenase

528 143 385 534 284 333 983

F: forward primers; R: reverse primers; Pax6: paired box 6; NF-L: neurofilament-low; NF-M: neurofilament-medium.

Fig. 1. Differentiation of cBMSCs and iBMSCs along osteogenic and adipogenic lineages. Alizarin red staining indicated mineral deposition in iBMSCs (A) and cBMSCs (B) cultured in osteogenic induction medium. Oil red-O staining indicated lipid clusters in iBMSCs (C) and cBMSCs (D) cultured in adipogenic induction medium. Scale bars, 100 ␮m.

(G3PDH) was used as a housekeeping gene. The sequences of the primers as well as the PCR conditions are shown in Table 1. 2.7. Western blot analysis Undifferentiated BMSCs and the cells induced to undergo neuronal differentiation for 10 days were examined for expression of NF-M. Cells were lysed with sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis sample buffer. Protein concentrations were measured with the BioRad protein assay (BIO-RAD Laboratories, Hercules, CA, USA). Proteins (20 ng) were separated on a 4.0% SDS-polyacrylamide stacking gel and 8.0% SDSpolyacrylamide separating gel, depending on molecular weight. Separated proteins were electroblotted onto a nitrocellulose mem-

brane (HybondTM-ECL, GE Healthcare, Little Chalfont, UK) and blocked with 5% bovine serum albumin overnight at 4 ◦ C. Immunodetection was performed with mouse mAbs against NF-M (1:1000, Cell Signaling Technology, Inc., Danvers, MA, USA) and detected with horseradish peroxidase-conjugated anti-mouse IgG (1:10000, Cell Signaling Technology, Inc.) as the secondary antibody. After washing, protein bands were detected with Pierce Western Blotting Substrate (Thermo Scientific, Waltham, MA, USA). 2.8. Immunostaining for a neuronal differentiation marker NF-M was examined as a neuronal differentiation marker. BMSCs were washed twice with PBS and fixed with 4% paraformaldehyde/1 M phosphate buffer for 30 min, and then non-

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Table 2 Percent of cells positive for cell surface markers with flow cytometry analysis. cBMSCs (n = 3) Percent positive

SD

iBMSCs (n = 3) Percent positive

SD

MSC markers 98.25 CD90 CD105 91.73 99.93 CD73

1.34 6.21 0.12

96.53 81.9 100

2.36 18.67 0

Endothelial/hematopoietic markers CD11b 2.6 CD34 2.9 CD45 2.6 2.95 HLA-DR

0.56 0.7 0.99 0.78

4.6 4.3 4.65 1.17

4.81 4.53 4.74 0.67

cBMSCs: cranial bone marrow mesenchymal stem cells; iBMSCs: iliac bone marrow mesenchymal stem cells; MSC: mesenchymal stem cell; HLA-DR: human leukocyte antigen-DR.

specific binding was blocked with 3% bovine serum albumin/PBS for 10 min at room temperature. NF-M (RMO14.9) mouse mAb (1:100; Cell Signaling Technology, Inc.) was incubated as the primary antibody overnight at 4 ◦ C, followed by Alexa Fluor 488-conjugated anti-mouse IgG antibody (1:500; Molecular Probes Europe BV Co., Leiden, NL) as a fluorescent secondary antibody for 60 min at room temperature. 4 ,6-diamidino-2-phenylindole dihydrochloride (DAPI; 1:1000; Kirkegaard & Perry Laboratories, Gaithersburg, MD, USA) was used to stain nuclei. Samples were mounted on glass slides with Vectashield® mounting medium for fluorescence (H-1000; Vector Laboratories, Burlingame, CA, USA). As a negative control, the primary antibody was omitted during immunostaining. Stained BMSCs were examined with a multifunctional microscope (BZ-9000; KEYENCE Co.). Images were stored in a computer for later analysis, and the percent of positive cells was calculated by dividing the number of positive cells by the total number of cells in 10 randomly selected fields of each sample. 2.9. Statistical analysis We analyzed the proportion of cells positive for neuronal markers between two different populations. Results are expressed as the means ± standard deviation. Statistical analyses were performed using the JMP statistical package (JMP version 7, SAS Institute Inc., Cary, NC, USA). To compare differences between two groups, we used the Mann–Whitney U-test for quantitative variables. A probability value of <0.05 was considered to be statistically significant. 3. Results 3.1. Flow cytometry analysis of specific cell markers Specific cell markers for cBMSCs and iBMSCs were characterized with flow cytometry analysis (Table 2). cBMSCs and iBMSCs showed similar characteristics. Both groups were strongly positive for CD90, CD105, and CD73 (cell surface markers associated with MSCs), but negative for CD11b, CD34, CD45, and HLA-DR (cell surface markers associated with endothelial/hematopoietic cells). 3.2. Multi-lineage differentiation capabilities To determine the ability to differentiate into multiple lineages, cBMSCs and iBMSCs were subjected to osteogenic and adipogenic differentiation. After osteogenic induction for 7 days, alizarin redpositive mineralized nodules were observed in both cBMSCs and iBMSCs (Fig. 1A, B). After 3 weeks of adipogenic induction, clusters of Oil red-O-positive cells were detected among the induced cBMSCs and iBMSCs (Fig. 1C, D).

Fig. 2. RT-PCR analysis of neural crest-associated genes expressed in both types of BMSCs (A) and the expression of neural genes before and after neural induction.(B). Expression of the neural crest-associated mRNAs p75, Slug, and Snail were detected in all cultures of cBMSCs (n = 3) and some cultures of iBMSCs (n = 3) (A). cBMSCs expressed these markers at remarkably higher levels than iBMSCs. Expression of the neural genes nestin (neural progenitor cell marker) and Pax6 (neural precursor marker) was detected in all cultures of cBMSCs (B). In iBMSCs, expression of nestin was lower than in cBMSCs, and Pax6 was not detected. After neural induction, cBMSCs expressed the neuronal markers NF-L and NF-M at remarkably stronger levels than iBMSCs. #2␮3#, “2”, and “3” are independent cultures established from different donors.

3.3. Neural crest-associated gene expression profile of BMSCs We used RT-PCR to evaluate neural crest-associated gene expression in cBMSCs and iBMSCs. All cultures of cBMSCs and some cultures of iBMSCs expressed p75, Slug, and Snail, which are associated with neural crest cells. cBMSCs expressed these markers remarkably stronger than iBMSCs (Fig. 2A). 3.4. Neural gene expression in BMSCs before and after neural induction To examine the neuronal differentiation capability, neural gene expression in cBMSCs and iBMSCs was analyzed with RT-PCR. The expression of nestin (neural progenitor cell marker) and Pax6 (neural precursor cell marker) was confirmed in cBMSCs before neurogenic induction. In iBMSCs, the expression of nestin was lower than in cBMSCs, and Pax6 was not detected. As seen with RT-PCR, cells differentiated from cBMSCs expressed the neuronal markers NF-L and NF-M at remarkably higher levels than cells differentiated from iBMSCs (Fig. 2B). 3.5. Neuronal protein marker expression following neural induction To confirm neuronal protein marker expression, NF-M expression in the induced BMSCs was evaluated with western blot

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Fig. 3. NF-M expression in differentiated BMSCs. Western blot analysis showed that differentiated cBMSCs expressed NF-M (A). “cBMSCs 1”, “cBMSCs 2”, and “cBMSCs 3” are independent cultures established from different donors. After neurogenic induction in both groups, immunofluorescence staining showed differentiated cells with scant cytoplasm and long processes that were positive for NF-M (B). The box plot shows the percentage of NF-M immunofluorescence-positive cells for each group (C, n = 9 per group). The boxes indicate the first quartile to the third quartile, and the lines in the boxes indicate the median. The error bars indicate minimum and maximum values. The number of NF-M-positive cells was significantly greater in groups of cBMSCs compared with groups of iBMSCs (P = 0.0003). Scale bars, 50 ␮m.

analysis. Differentiated cBMSCs expressed NF-M, but no expression was detected in differentiated iBMSCs (Fig. 3A).

3.6. Immunostaining for a neuronal differentiation marker To examine the neurogenic potential of cBMSCs and iBMSCs, expression of NF-M protein in differentiated BMSCs from each cell group was investigated with immunostaining. After neural differentiation in both groups, we detected cells with scant cytoplasm and long processes that were positive for NF-M (Fig. 3B). The percent of cells positive for NF-M in cBMSCs was 27.1 ± 7.0% (mean ± SD), which was significantly higher than that of iBMSCs (10.1 ± 3.2%) (n = 9, P = 0.0003, Fig. 3C).

4. Discussion Today, clinical cell therapies using MSCs for CNS disorders are being developed, and they require a source of donor cells with more efficient neuronal differentiation capabilities. In the present study, we isolated an MSC population from human cranial bone marrow and confirmed that cBMSCs have a tendency to differentiate into neuron-like cells. No report has analyzed the characteristics of human cBMSCs. The research and investigation of cell transdifferentiation have also majorly developed in recent decades. Depending on the manipulation, differentiated cells are able to transdifferentiate to another cell type across lineage boundaries. However, dedifferentiation in the process of transdifferentiation has a potential problem of neoplastic transformation. Therefore, MSCs that have multipotency without requiring dedifferentiation should be easier to use for cell therapy.

To be defined as MSCs, cells must be capable of differentiating into multiple lineages [10]. After osteogenic and adipogenic induction, Alizarin red-positive mineralized nodules and Oil red-Opositive lipid droplets were observed in respective cBMSC cultures and showed a similar appearance as iBMSCs (Fig. 1), demonstrating the multipotential characteristics of both cell types. These MSC characteristics of both cell types were confirmed with flow cytometry analysis of the cell surface marker phenotype of the cells (Table 2). These data suggest that our initial cell isolation and primary culture procedures were performed rigorously and that we successfully established primary cell populations of cBMSCs that were not contaminated with hematopoietic cells. Sakai et al. reported that tooth-derived stem cells are derived from cranial neural crest cells and have unique neuroregenerative activities that are not exhibited by any other previously described stem cells. They described that tooth-derived stem cells may provide significant therapeutic benefits for treating the acute phase of spinal cord injury through both cell-autonomous and paracrine/trophic regenerative activities [11]. In an analogous way, cBMSCs, which are also derived from the neural crest, are likely to have high neurogenic potential because they have the same origin as neurons and glial cells. Our cBMSCs expressed mRNAs associated with neural crest cells in RT-PCR, and the expression was higher than that in iBMSCs (Fig. 2A). To investigate the neurogenic potential of our cBMSCs, neurogenic differentiation was induced in the cultures. RT-PCR analysis showed that expression of neuronal markers in cBMSCs was remarkably higher than expression in iBMSCs. Similarly, western blot analysis showed high expression of the neural protein marker NF-M in differentiated cBMSCs, although differentiated iBMSCs showed undetectable NF-M expression. NF-M expression in differentiated cBMSCs was also observed with immunofluorescence staining. Moreover, we quantitatively assessed neurogenic changes

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by counting NF-M-positive cells. The results showed a significantly greater proportion of NF-M-positive cells within the population of induced cBMSCs compared with the population of iBMSCs. These findings suggested that our established cBMSCs have higher neurogenic potential than standard MSCs and may be useful for stem cell research and stem cell therapy for neural disease. However, whether cBMSCs will actually facilitate functional recovery after CNS dysfunction remains unknown. Further investigation of the effects following cBMSC transplantation in animal experiments or clinical studies will be required. We have described the establishment and characterization of MSCs derived from cranial bone marrow and compared these cells with iBMSCs, which are considered standard BMSCs. Both populations of BMSCs showed almost identical properties regarding multipotency traits. However, the two populations varied in their gene expression profiles and neurogenic differentiation tendency. The property that cBMSCs are better able to differentiate into the neurogenic lineage and our ability to obtain cBMSCs from what is essentially medical waste after routine neurosurgical procedures represent advantages of cBMSCs compared with BMSCs obtained from the bones in other places in the body, the collection of which requires additional invasive procedures. We believe that cBMSCs are a favorable source of donor cells for autologous transplantation for CNS regenerative therapy. Our next experiment will be establishment of cBMSCs using autologous human serum or synthetic media without xenogeneic reagents to obtain safer donor cells for human clinical use. The concept of cBMSC-based medicine may facilitate development of cell therapies for treating CNS disorders such as stroke, brain/spinal cord injury, and neurodegenerative disease. 5. Conclusions Isolation and establishment of cBMSCs from human cranial bone wastes have been achieved. Our established cBMSCs were confirmed that these have multilineage differentiation potential and specific cell markers required to be defined as MSCs. In addition, these showed a greater tendency to differentiate into neuron-like cells than iBMSCs. Therefore, cBMSCs would be considered as efficient source of donor cells for CNS regenerative medicine.

Competing interests The authors declare no conflicts of interest. Acknowledgements This work was supported in part by a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (JSPS KAKENHI grant number 23592127). References [1] F.H. Gage, Mammalian neural stem cells, Science 287 (2000) 1433–1438. [2] A. Björklund, O. Lindvall, Self-repair in the brain, Nature 405 (2000), 892-3, 895. [3] S. Temple, Division and differentiation of isolated CNS blast cells in microculture, Nature 340 (1989) 471–473. [4] O. Honmou, K. Houkin, T. Matsunaga, Y. Niitsu, S. Ishiai, R. Onodera, S.G. Waxman, J.D. Kocsis, Intravenous administration of auto serum-expanded autologous mesenchymal stem cells in stroke, Brain 134 (2011) 1790–1807. [5] H. Liu, O. Honmou, K. Harada, K. Nakamura, K. Houkin, H. Hamada, J.D. Kocsis, Neuroprotection by PlGF gene-modified human mesenchymal stem cells after cerebral ischaemia, Brain 129 (2006) 2734–2745. [6] T.W. Sadler, Langman’s Medical Embryology, twelfth edition, Lippincott Williams and Wilkins, Philadelphia, 2012. [7] Y. Tamaki, T. Nakahara, H. Ishikawa, S. Sato, In vitro analysis of mesenchymal stem cells derived from human teeth and bone marrow, Odontology 101 (2013) 121–132. [8] S.T. Lee, J.H. Jang, J.W. Cheong, J.S. Kim, H.Y. Maemg, J.S. Hahn, Y.W. Ko, Y.H. Min, Treatment of high-risk acute myelogenous leukaemia by myeloablative chemoradiotherapy followed by co-infusion of T cell-depleted haematopoietic stem cells and culture-expanded marrow mesenchymal stem cells from a related donor with one fully mismatched human leucocyte antigen haplotype, Br. J. Haematol. 118 (2002) 1128–1131. [9] S. Suon, H. Jin, A.E. Donaldson, E.J. Caterson, R.S. Tuan, G. Deschennes, C. Marshall, L. Iacovitti, Transient differentiation of adult human bone marrow cells into neuron-like cells in culture: development of morphological and biochemical traits is mediated by different molecular mechanisms, Stem Cells Dev. 13 (2004) 625–635. [10] E.M. Horwitz, B. Le, K. lanc, M. Dominici, I. Mueller, I. Slaper-Cortenbach, F.C. Marini, R.J. Deans, D.S. Krause, A. Keating, Clarification of the nomenclature for MSC: the International Society for Cellular Therapy position statement, Cytotherapy 7 (2005) 393–395. [11] K. Sakai, A. Yamamoto, K. Matsubara, S. Nakamura, M. Naruse, M. Yamagata, K. Sakamoto, R. Tauchi, N. Wakao, S. Imagama, H. Hibi, K. Kadomatsu, N. Ishiguro, M. Ueda, Human dental pulp-derived stem cells promote locomotor recovery after complete transection of the rat spinal cord by multiple neuro-regenerative mechanisms, J. Clin. Invest. 122 (2012) 80–90.