Abstract



BACKGROUND: It has been widely accepted that both bone marrow-derived mesenchymal stem cells (MSCs) and hematopoietic stem cells (HSCs) have the capacity to differentiate into neural lineages. Some scholars believe that in addition to HSCs and MSCs, bone marrow (BM) also harbors a highly mobile population of CXCR4+ tissue committed stem cells (TCSCs), including skeletal muscles, heart, liver, and neural tissue.
OBJECTIVE: To make sure that neural tissue-committed stem cells (NTCSCs) reside in the bone marrow, and to establish a purification and culture method for bone marrow-derived NTCSCs.
DESIGN: Opening animal study.
SETTING: Department of Anatomy, the Second Military Medical University of Chinese PLA.
MATERIALS: Adult Sprague-Dawley (SD) rats (pathogen-free) were provided by the Animal Center of the Second Military Medical University of Chinese PLA. Dulbecco's modified eagle's medium (DMEM)/F12, B27, N2 and epidermal growth factor (EGF, Invitrogen Company), basic fibroblast growth factor (bFGF, CytoLab Ltd), rabbit anti-rat Nestin,CXCR4, β-Tublin Ⅲ, glial fibrillary acidic protein (GFAP, Santa Cruz Company), mouse anti-rat microtubule associated protein 2ab (MAP2ab) (Clone11-5B), cyclic nucleotide 3'phosphohydrolase （CNPase, Clone AP20, NeoMarkers Company）, fluorescent(fluorescein isothiocyanate, Cy3) marker reagents (Wuhan Boster Bioengineering Co., Ltd), nuclear fluorescent dyes 4,6-diamidino-2-phenylindole(DAPI)(Sigma), immunohistochemistry reagents (Vector Laboratories Company) , and NycoPrepTM separation liquid (1.077A, Axis-Shield Company) were used in this study.
METHODS: This study was performed in the Department of Anatomy, the Second Military Medical University of Chinese PLA from January 2004 to December 2006. Bone marrow was harvested from bilateral femurs and tibias of 2-3 weeks SD rats. Mononuclear cell layer was isolated by NycoPrepTM separation liquid and suspended in DMEM/F12(1:1)serum-free medium supplemented with 2% B27,1% N2, 20 μg/L bFGF, 20μg/L EGF, 1×105 U/L penicillin and 100 mg/L streptomycin. NTCSCs were isolated and propagated by suspensive growing from adherent cells in bone marrow in DMEM/F12 free-serum medium.
MAIN OUTCOME MEASURES: NTCSCs were identified by immunocytochemistry for CXCR4, a marker of TCSCs and nestin, a marker of neural stem cells, and neural lineages marker protein after differentiation of cellular spheres.
RESULTS: The NTCSCs spheres expressed nestin, a neural stem cell marker as well as CXCR4, a marker of TCSCs. The NTCSCs' spheres were naturally differentiated in DMEM medium with 15% fetal bovine serum. The differentiated cells expressed β-Tublin Ⅲ, MAP2ab, CNPase and GFAP, markers of neural lineages.
CONCLUSION: NTCSCs reside in bone marrow and naturally differentiate into neural lineages in vitro.

INTRODUCTION

Bone marrow contains two major cell types, hematopoietic stem cells (HSCs) and mesenchymal stem cells (MSCs). Several reported experiments have demonstrated that MSCs and HSCs have cross-mesoderm differentiation potential and can transdifferentiate into neural cells in vitro [1-3]. Researches [4-9] of HSCs and MSCs transplantation in models of animal also have shown that they can reduce the infarct scope and promote the neural function after HSCs and MSCs transplantation into brain and spinal cord. Furthermore, part of transplanted cells expressed neuron specific enolase (NSE), microtubule associated protein 2ab (MAP2ab), neurofilament (NF), glial fibrillary acidic protein (GFAP), etc. The concept of so-called plasticity or trans-dedifferentiation of HSCs and MSCs has recently been called into questions [10]. Kucia et al [11-13] thought that in addition to HSC and MSCs, bone marrow (BM) also harbored a highly mobile population of CXCR4+ tissue committed stem cells (TCSCs), including skeletal muscles, heart, liver, and neural tissue, which can differentiate into muscle, cardiac, liver and neural cells. We have done the study to make sure that neural tissue-committed stem cells (NTCSCs) reside in bone marrow and to create a culture and purification method for bone marrow-derived NTCSCs. A new method of NTCSCs isolation and culture was provided with brief manipulation and low-cost. Transplanting cells provide a therapy for neurological diseases and traumatic repair in brain and spinal cord, which are derived from autologous bone marrow with wide clinical application, no immune rejection, and no ethical or moral problems.

MATERIALS AND METHODS

Materials
This study was performed in the Department of Anatomy, the Second Military Medical University of Chinese PLA from January 2004 to Devember 2006. Twenty-eight adult male Sprague-Dawley (SD) rats of pathogen-free, aged 3-4 weeks, weighing （80±20）g, were provided by the Animal Center of the Second Military Medical University of Chinese PLA[Production permit No. SCXK (Shanghai) 2002-2006; use permit No. SYXK (Shanghai) 2002-2006]. The experimental procedures were conducted in accordance with the

animal care guidelines of the Institute of Health.
Main reagent: Dulbecco's modified eagle's medium (DMEM)/F12, B27, N2 and epidermal growth factor ( EGF, Invitrogen Company), basic fibroblast growth factor ( bFGF, CytoLab Ltd), rabbit anti-rat Nestin,CXCR4, β-Tublin Ⅲ, glial fibrillary acidic protein (GFAP, Santa Cruz Company); mouse anti-rat microtubule associated protein 2ab (MAP2ab)（Clone11-5B）, cyclic nucleotide 3'phosphohydrolase (CNPase), (Clone AP20, NeoMarkers Company), fluorescent(fluorescein isothiocyanate, Cy3) marker reagents (Wuhan Boster Bioengineering Co.,Ltd), nuclear fluorescent dyes 4,6-diamidino-2-phenylindole (DAPI) (Sigma), immunohistochemistry reagents (Vector Laboratories Company) , and NycoPrepTM separation liquid (1.077A, Axis-Shield Company) were used in this study.

Methods
Preparation of bone marrow cell suspension
Bone marrow was harvested from bilateral femurs and tibias of 2-3 weeks old SD rats. Mononuclear cell layer was isolated by NycoPrepTM separation liquid and suspended in DMEM/F12(1:1)serum-free medium supplemented with 2% B27, 1%N2, 20 μg/L bFGF, 20 μg/L EGF, 100 U/mL penicillin and 100 mg/L streptomycin. NTCSCs were isolated and propagated by suspensive growing from adherent cells in bone marrow in DMEM/F12 free-serum medium.

Cultivation and purification of bone marrow-derived NTCSCs
The cell suspension (cell density 5×107 L-1) was seeded in a 25 cm2 flask coated with poly-L-ornithine. The cells were incubated in a 5% CO2-air humidified chamber at 37 ℃ for 48 hours. Non-adherent cells were then removed by changing medium. The adherent cells were transferred to dishes coated with poly-L-ornithine and fibronectin at a concentration of 2.0×106 L-1. The cells were cultured adherently for 2-4 weeks and half medium was renewed every three days. A group of cluster-like cells were selected to make cell suspension. The cell suspension (cell density 1-2/well) was seeded onto a 96-well plate coated with poly-L-ornithine and fibronectin. After colony formation, the cells were cultured in a 25 cm2 flask. Cell spheres were observed for 4-5 days. After a large number of cell spheres formed, they were suspended and propagated at 1:2 dilution.

Identification of NTCSCs
Immunohistochemical staining of NTCSCs from marrow: Cell spheres were selected and dripped onto the coverslip by a glass straw. The cell spheres were fixed in 4% paraformaldehyde , 0.1 mol/L phosphate buffer solution（pH 7.4）for 10 minutes, immerged into 0.2% Triton X-100 /Tris-solution for 15 minutes, and incubated with sheep serum for 30 minutes, added to nestin antibody (1:200) and CXCR4 antibody（1:200）, and incubated overnight at 4 ℃. Secondary antibody was biotin-labeled anti-rabbit IgG(1:200) and incubated at room temperature. ABC complexes were incubated at room temperature for 40 minutes. The suspension was rinsed with 0.01 mol/L phosphate buffer solution after each step. Then, nestin and CXCR4 were respectively detected by alkaline phosphatase and DAB staining, and observed under a microscope.
Immunofluorescent staining of NTCSCs from bone marrow: The formed cell spheres were selected and dripped onto the coverslip by a glass straw. The cell spheres were fixed in 4% paraformaldehyde, 0.1mol/L phosphate buffer solution（pH 7.4）for 10 minutes, and immerged into 0.2% Triton X-100 /Tris-solution for 15 minutes, incubated with sheep serum for 30 minutes, then added respectively to nestin antibody (1:200), CXCR4 antibody (1:200) overnight at 4℃. Nestin and CXCR4 were detected respectively by goat anti-rabbit IgG-Cy3 (1:50, red fluorescence) and goat anti-rabbit FITC (1:50, green fluorescence), and incubated at room temperature away from the light. The suspension was rinsed with 0.01 mol/L phosphate buffer solution after each step and observed under a fluorescence microscope.
Natural differentiation of NTCSCs of bone marrow: The cells spheres were dripped onto a culture plate. They were incubated in the DMEM supplemented with 15% fetal bovine serum. One week later, marker protein of neurons and glial cells: MAP2ab (mouse anti-rat,1:100), β-Tublin Ⅲ(rabbit anti-rat,1:100), GFAP (rabbit anti-rat, 1:100), CNPase (mouse anti-rat, 1:100), etc., were detected. The secondary antibody was detected by goat anti-rabbit FITC (1:50, green fluorescence). The nucleus was stained by diamidino-phenyl-indole (DAPI).

RESULTS

Observation of primarily cultured cells and colony formation
The cells were seeded onto 6-well plates coated with poly-L-ornithine and fibronectin. Afterwards, they were observed according to growing spherically and adherently with a character of easily reflecting light under an inverted microscope. Two days later, part of cells died, presenting a slag-like material. However, other part of cells enlarged with enhanced reflection, presented division phase, and formed cell clusters including 2-5 cells (Figure 1). Cell clusters increased and enlarged, and 21 days later, they formed bigger and more cell clusters containing dozens of cells (Figure 1). The screened cells were transferred into a culture flask. Five or six days later, cell spheres formed and contained 3-4 or dozens of cells together (Figure 1).

Identification of bone marrow-derived NTCSCs
By immunohistochemical and immunofluorescent detections, the cell spheres expressed nestin, a marker protein of neural stem cell and CXCR4, a marker protein of TCSCs. Immunohistochemical staining showed that nestin was blue, and CXCR4 was brown (Figure 2). Immunofluorescent staining showed the former was red and the latter was green (Figure 3).

Differentiation of bone marrow-derived NTCSCs
Cell spheres adhered to the flask plate in DMEM supplemented with 15% fetal bovine serum for 30 minutes. After 24-hour culture, external cells of the spheres differentiated with 2-3 projections, and part of cells migrated outward (Figure 1d). Seven days later, differentiated cells presented with reticular distribution, multiple angle and spindle shaped. By immunofluorescent detection, the cells expressed β-Tublin Ⅲ, MAP2ab, CNPase, GFAP, and etc. Cytoplasm presented with green fluorescence and re-stained nucleus was blue. β-Tublin Ⅲ-postive,MAP2ab-postive cell body was relatively larger, with 2-5 thick projections. CNPase-postive and GFAP-postive cell body was smaller with thin projections (Figure 4).

DISCUSSION

In 2002, Kabos et al [14] reported that cell spheres were separated from rat bone marrow and cultured, and were compared with forebrain-derived nerve spheres. Both of them have no significant differences in morphology. Bone marrow-derived cell spheres also express nestin, vimentin protein, and tissue-special transcription factors （neurogenin1） in the neural development period. Bone marrow-derived cell spheres have similar differentiation-characteristics to brain-derived cellular spheres. In addition, the cells differentiated by the former express neuron marker protein NeuN, NSE, MAP2 and NF-200, as well as astrocyte marker protein GFAP and oligodendrocyte marker protein CNPase. Magaki et al [15] successfully cultured neural stem cell-like cell spheres from bone marrow by a monolayer adherent serum-free culture method. This data confirmed the study of Kabos et al. In 2006, Kucia et al [13] screened NTCSCs from mouse bone marrow using density centrifugation, immunobeads, and fluorescence activated cell sorter, which can form cellular spheres in vitro. Further studies confirmed that NTCSCs in bone marrow could be released into blood when brain stroke occurs. Though Kabos et al and Magaki et al did not thoroughly study the cell source of cellular spheres, we guess these cell spheres are derived from NTCSCs. Moreover, they should have similar biological growing characteristics to neural stem cells. Finally, serum-free culture liquid for neural stem cells is also suitable for cultivation of NTCSCs. We initially used the method of adhesion, and then used suspension with serum-free culture liquid for neural stem cells to separate and culture monoclonal NTCSCs. By immunohistochemical and immunofluorescent detections of cell spheres, as well as detection of neuron and glial cells marker after cell spheres spontaneous differentiation, our cell spheres had the characteristics of neural stem spheres. Tissue-committed stem cells marker protein CXCR4 was positive for cell spheres, which had the similar characteristics to separated NTCSCs by Kucia et al [13]. These were NTCSCs derived from bone marrow. As seed cells, NTCSCs provide a wide application prospect in the treatment of nervous system diseases concerning brain and spinal cord.

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骨髓中一类新细胞群——神经组织定向
干细胞的确立☆

张志英，任丛莉，张传森，李 亮，党瑞山，孔征东
解放军第二军医大学解剖教研室，上海市 200433
张志英☆，女，1963年生，山西省平遥县人，汉族，2002年上海中医药大学毕业，博士，副教授，主要从事神经再生与修复的研究。
通讯作者：张传森, 博士，解放军第二军医大学解剖教研室，上海市 200433
摘要
背景：骨髓间充质干细胞和造血干细胞具有分化为神经类细胞的潜能已得到共识。另有研究者认为骨髓中除骨髓间充质干细胞和造血干细胞以外，还寄居着CXCR4阳性的组织定向干细胞，包括骨骼肌、心、肝及神经组织定向干细胞。
目的：实验拟进一步证实神经组织定向干细胞寄居于骨髓，并寻求确立神经组织定向干细胞分离、培养的新方法。
设计：开放性实验。
单位：解放军第二军医大学解剖学教研室。
材料：所用成年清洁级SD大鼠由解放军第二军医大学动物中心提供。DMEM/F12，B27，N2均购自Invitrogen公司；bFGF源于CytoLab Ltd；EGF源于Invitrogen公司；Nestin、CXCR4，β-Tublin Ⅲ，神经胶质纤维酸性蛋白等一抗为Santa Cruz公司兔抗鼠多克隆抗体；MAP2ab（Clone11-5B），CNPase（Clone AP20）为NeoMarkers公司小鼠抗大鼠单克隆抗体；荧光（FITC，Cy3）标记试剂盒购自武汉博士德生物工程有限公司；免疫组化试剂盒购自Vector Laboratories公司。NycoPrepTM分离液（1.077A）购自Axis-Shield 公司。
方法：实验于2004-01/2006-12在解放军第二军医大学组织工程研究所完成。取SD大鼠双侧股骨及胫骨，冲洗骨髓腔，经NycoPrepTM分离液分离单核细胞层，DMEM/F12（1∶1） 无血清神经干细胞培养液（内含2%B27，1%N2，20 μg/L碱性成纤维细胞生长因子， 20 μg/L 表皮生长因子,1×105 U/L青霉素, 100 mg/L链霉素），通过先贴壁、后悬浮的方法从骨髓中分离、培养单克隆生长的神经组织定向干细胞。
主要观察指标：通过免疫细胞化学法检测神经组织定向干细胞细胞球CXCR4和nestin蛋白，以及细胞球自然分化后神经元以及神经胶质细胞的标志蛋白。
结果：①神经组织定向干细胞细胞球表达神经干细胞标志蛋白nestin和组织定向干细胞标志蛋白 CXCR4。②神经组织定向干细胞细胞球在含15%胎牛血清的DMEM培养液中可自然分化，分化细胞呈梭形或多角形，有2~5个细胞突起，免疫荧光检测显示神经元标志蛋白β-tublinIII及MAP2ab，以及神经胶质标志蛋白CNPase和神经胶质纤维酸性蛋白阳性。
结论：骨髓中存在神经组织定向干细胞，可自然分化为神经元样细胞及神经胶质样细胞。
关键词：骨髓；神经组织定向干细胞；细胞培养；鉴定；细胞移植
中图分类号: R394.2 文献标识码: A 文章编号: 1673-8225(2008)08-01577-04
张志英，任丛莉，张传森，李亮，党瑞山，孔征东.骨髓中一类新细胞群——神经组织定向干细胞的确立[J].中国组织工程研究与临床康复，2008，12(8):1577-1580
[www.zglckf.com/zglckf/ejournal/upfiles/08-8/8k-1577(ps).pdf]
（Edited by Martin F/Song LP/Wang L）