Abstract
BACKGROUND: It has been reported that in China, human bone marrow mesenchymal stem cells are mostly harvested from adults. Studies on bone marrow mesenchymal stem cells in children are few.
OBJECTIVE: To isolate and expand bone marrow mesenchymal stem cells from children, and to analyze the biological characteristics of bone marrow mesenchymal stem cells and their potential of differentiating into osteoblasts, adipocytes and neural like cells.
DESIGN: Observational comparative study.
SETTING: Tongji Medical College, Huazhong University of Science and Technology.
MATERIALS: Experiments were performed at the Laboratory of Department of Orthopaedics of Wuhan Tongji Hospital from March to September 2006. Bone marrow mesenchymal stem cells were collected from one boy patient and two girl patients aged 5-8 years, who received pelvis osteotomy for dysplasia of the hip joint. The experimental procedures were approved by the Hospital Ethics Committee and family members of all children patients singed the informed consent. Dexamethasone, vitamin C, β-sodium glycerophosphate, 3-Isobutyl-1-methylxanthine, insulin, indometacin and butylated hydroxyanisole were bought from Sigma Company. Dimethyl sulphoxide was purchased from Amersco Company.
METHODS: Bone marrow mesenchymal stem cells were cultured from mononuclear cells isolated over a Percoll gradient. Bone marrow mesenchymal stem cells were observed under an inverted phase contrast microscope. Bone marrow mesenchymal stem cells could differentiate into osteoblasts, adipocytes and neural like cells with osteoblast inductor (β-sodium glycerophosphate, dexamethasone, vitamin C), lipoblast inductor (dexamethasone, 3-isobutyl-1-methylxanthine, bovine insulin, indometacin) and serum-free medium inductor (dimethyl sulphoxide, butylated hydroxyanisole) respectively. Osteoblast marker (alkaline phosphatase, osteocalcin mRNA, calcium node), adipocyte marker (lipid droplet, PPAR γ-2mRNA) and neural cell-like marker (nissl body, neuron specific enolase, neurofilament protein) were respectively determined by the immunohistochemical method, polymerase chain reaction and immunocytochemical method.
MAIN OUTCOME MEASURES: ①Appearance and proliferation of bone marrow mesenchymal stem cells from children, and ②determination results of osteoblast, adipocyte and neural cell markers.
RESULTS: ①Children bone marrow mesenchymal stem cells could easily adhere to the wall, appeared fusiform, had high reproductive activity and arranged vortically after fusing. ②Appearance of bone marrow mesenchymal stem cells changed after receiving inductor. Osteoblast marker, adipocyte marker and neural cell-like marker were positive after chemical staining, polumerase chain reaction and immunocyte staining.
CONCLUSION: Children bone marrow mesenchymal stem cells show stable proliferation, passage and multi-direction differentiation towards osteoblasts, adipocytes and neural like cells.

INTRODUCTION

Bone marrow mesenchymal stem cells (BMSCs) are characterized by easily collecting materials, in vitro isolation, culture, amplification, and no immunological rejection after autotransfusion, high gene transfection efficiency and stability after transfection. Thus, BMSCs are considered as important cell population for use in tissue engineering and gene engineering[1-3]. In China, BMSCs are mostly harvested from adults, and studies on BMSCs from children are few. This article aims to isolate and culture BMSCs from children, and to observe the biological characteristics and the potential in differentiating into osteoblasts, adipocytes and neural like cells.

MATERIALS AND METHODS

Materials
Experiments were performed at Laboratory of Department of Orthopaedics of Wuhan Tongji Hospital from March to September 2006. All the authors did the design, data collection, intervention and evaluation. Persons above mentioned had done the strict training on empirical method. BMSCs from children were collected from one boy patient and two girl patients aged 5-8 years, who received pelvis osteotomy for dysplasia of hip joint. The experimental procedures were approved by Hospital Ethics Committee. Family members or leagal guardians of all children patients singed the informed consent.
Main reagents included DMEM medium (America Hyclone Company), fetal bovine serum (Sijiqing Company), MTT, dexamethasone, vitamin C, β-sodium glycerophosphate, 3-isobutyl-1-methylxanthine, insulin, indometacin, β-mercaptoethanol and butylated hydroxyanisole (Sigma Company), Trypase and dimethyl sulphoxide (Amersco Company), Cobaltous nitrate, ammonium sulfide, natrium thiosulfuricum and argentums nitricum (Beijing Chemical Agent Factory).Primary antibodies of neurone specific enolase and neurofilament-200 protein were presented by Department of Pathology of Tongji Medical College.

Osteocalcin and PPARγ-2 were provided by Shanghai Yingjun Gene Company.
Main instruments: Inverted phase contrast microscope, Enzyme-linked Immunosorbent Meter of DG3022A type, ultraviolet spectrophotometer and refrigerated centrifuge were respectively purchased from Japan OLYMPUS, Huadong Electron Tube Factory, Japan Daojin (1240) and America Eppendorf.

METHODS
Cell collection and culture: 4-5 mL of bone marrow was collected from the iliac crest of patients, who received 100 000 U/L anticoagulation with heparin. BMSCs were isolated by Percoll lymph-separating medium density gradient method, and then incubated in L-DMEM plus 10% fetal bovine serum plus double antibodies (100 000 U/L penicillin and 100 000 U/L streptomycin) in incubator containing CO2 of 0.05 volume fraction at 37 ℃ and saturated humidity. Culture fluid was regularly changed for passage. BMSCs were purified by repetitive adherence. The appearance of BMSCs was observed under an inverted phase contrast microscope.
Determination of cell proliferation and the drawing of growth curve:Second generation of BMSCs were harvested and made into cell suspensions, and then incubated in seven 96-well plates at the same concentration, 4 wells in each plate, 200 μL cell suspension in each well, and then put into incubator containing CO2 of 0.05 volume fraction at 37 ℃. Culture fluid was changed every 3 days. From the 2nd day, 5 g/L MTT of 20 μL was added every day for successively 7 days, misce bene. Four hours later, culture fluid was removed, and 150 μL DMSO was added and swung for 10 minutes at room temperature. Absorbance of 4 wells was measured at 490 nm of enzyme-labeling reader. Results were expressed as Mean±SD. Growth curve was drawn according to the absorbance.
Determination of cell cycle: Third passage of bone marrow mesenchymal stem cells were incubated in 6-well plates at 5×107 L-1. BMSCs were stained with propiolium iodide for 30 minutes. Cell cycle was measured with a flow cytometer.
Differentiation of BMSCs into osteoblasts: Third to fifth passaged BMSCs were collected, digested with 0.25% pancreatin, blew into cell suspensions, and incubated in 6-well plate. These BMSCs were divided into 2 groups when covered 80% the surface of the culture plate. BMSCs in the induction group were incubated in the medium of inductor (10 mmol/Lβ-sodium glycerophosphate, 10-8 mmol/L dexamethasone, 50 mg/L vitamin C) containing osteoblasts. BMSCs in the control group were incubated in the medium of inductor without osteoblasts. Culture fluid was changed every 3 days. Two weeks later, alkaline phosphatase levels were measured by Gomori calcium-cobalt method. Three weeks later, lysed cells were detected by reverse transcription polymerase chain reaction to determine osteocalcin gene (Primer: positive-sense strand 5' ATG AGA GCC CTC ACA CTC CTC 3', antisense strand 5' CTA GAC CGG GCC GTA GAA GCG 3', polymerase chain reaction product of 303 bp, reannealing at 55 ℃). Four weeks later, Von Kossa was used to determine calcium nodus.
Differentiation of BMSCs into adipocytes: Preparation was the same as that mentioned above. BMSCs in the induction group were incubated in the high glucose medium of inductor (1 μmol/L dexamethasone，0.5 mmol/L 3-Isobutyl-1-methylxanthine, 10 mg/L bovine insulin, 100 mmol/L indometacin) containing adipocytes. Bone marrow mesenchymal stem cells in the control group were incubated in the low glucose medium of inductor without adipocytes. Culture fluid was changed every 3 days. Two weeks later, fatty oil drop was identified by Oil Red O Method. Three weeks later, lysed cells were detected by reverse transcription polymerase chain reaction to determine PPARγ-2 gene (Primer：positive-sense strand 5' CAT TCT GGC CCA CCA ACT T 3'，antisense strand 5' CCT TGC ATC CTT CAC AAG CA 3'，polymerase chain reaction product of 372 bp，reannealing at 55 ℃).
Differentiation of BMSCs into neural cells: Preparation was the same as that mentioned above. BMSCs in the induction group were induced with 20% fetal bovine serum, 3 mmol/Lβ-
mercaptoethanol for 24 hours, washed with PBS three times, and then induced with serum-free medium containing 20 g/L dimethyl sulphoxide and 200 mmol/L butylated hydroxyanisole. BMSCs in the control group were incubated with serum medium. Six hours later, nissl body of BMSCs was observed by toluidine blue staining. Neuron specific enolase and neurofilament-200 protein levels were determined by immunohistochemical method.

RESULTS

Isolation, culture and proliferation of BMSCs
Primarily incubated BMSCs were round, large, lucency, had strong refraction. Nuclei were oval and mixed with some blood cells. 2-4 days after adherence, BMSCs were fusiform. 3-4 days later, BMSCs rapidly proliferated and showed clone cell-like. 12-14 days later, cell appearance was more similar when 80% confluence was achieved. BMSCs grew rapidly after passaging. 7-10 days later, BMSCs could adhere to the whole bottom of culture flask, arrange in swirl till confluence. The proliferation of BMSCs of the second generation was determined by MTT method. Growth curve was S-shape (Figure 1). 2-3 days later, BMSCs entered exponential growth phase, and 7-9 days later, entered platform growth phase.

Determination of cell cycle
It is found that （89.35±0.92）% BMSCs of the 3rd generation from children were in G0/G1 phase, （4.85±0.46）% in S phase, and （5.80±1.31）% in G2/M phase. Proliferation index（S+G2/M）was 10.65±0.92 (Figure 2).

BMSCs after inducing with osteoblast inductor
After inducing with osteoblast inductor, BMSCs had significant changes in appearance. From the 3rd day after induction, some BMSCs changed from fusiform to three-dimensional, became larger, and changed into polygon. With the prolongation of time, triangle or polygonal cells became more, and then grew in multilayers; many crystal particles were seen. Alkaline phosphatase level was determined by Gomori Ca-Co method. It was found that brown-black particles appeared in cytoplasm, which was significant as compared with the control group (Figure 3). Osteocalcin gene was measured by reverse transcription polymerase chain reaction. Osteocalcin mRNA was obviously reinforced in the induction group (Figure 4). Calcium nodus was determined by Von Kossa staining. It was shown that calcification nodus was found among cells in the induction group (Figure 5).

BMSCs after inducing with adipocyte inductor
After inducing with adipocyte inductor, BMSCs were round or nearly round, arranged in whirl, and then became irregular; cell proliferation was inhibited; Tiny lipid droplets appeared in cytoplasm that became larger, lucency, with strong refraction; Nuclei were in one side of cells. Two weeks after induction, about 60% BMSCs became adipocyte-like. Lipid droplets were red after Oil red O staining. Three weeks later, PPARγ-2 was positive, but it was negative in the control group after reverse transcription polymerase chain reaction (Figure 6).

BMSCs after inducing with neural cell inductor
Twenty-four hours after pre-intervening with neural cell inductor, BMSCs were crenation, irregular, and many slender processes appeared at the fringe of cells (Figure 7a). After formal induction, some BMSCs were conical, like axon structure. 6 hours later, the appearance of cell was stable (Figure 7b). Toluidine blue stain showed that nissl body appeared in most BMSCs after fixing with 10% formalin. (Figure 8); It was found that the neurone specific enolase and neurofilament-200 protein were positive (Figure 9).

DISCUSSION

Multi-directional differentiation had been considered as the most important biological characteristic of BMSCs, since Friedenstein et al successfully cultured BMSCs in 1970s. In this study, we successfully induced the differentiation of BMSCs into osteoblasts with the combination of vitamin C, β-sodium glycerophosphate and low concentration of dexamethasone. Vitamin C can accelerate the collagen synthesis and calcification in cells, adjust adenosine triphosphate and alkaline phosphatase levels as well as the synthesis of non-collagenous matrix protein. Beta sodium glycerophosphate could make phosphorus ions as the substrate of alkaline phosphatase and accelerate the deposition and calcification of calcium salts[4]. Dexamethasone belonged to glucocorticoid could accelerate an increase in alkaline phosphatase levels of BMSCs and the differentiation of BMSCs into osteoblasts; it could mainly accelerate matrix synthesis in an earlier period, but calcification in later period[5]. Alkaline phosphatase is the early marker enzyme during the differentiation into osteoblasts. Hydrolysis phosphate esters can offer necessary phosphoric acid for the deposition of hydroxyapatite ceramic. Osteocalcin that is the mature marker of the differentiation into osteoblasts mainly can maintain the normal mineralization rate of the bone, prevent the formation of abnormal hydroxylapatite crystal, and accelerate the normal calcification of mineral matter deposition of bone tissue. The formation of bone node is the main marker of in vitro osteogenesis, and the bone node is formed by accumulation of the matrix, deposition and confluence of rock salt[6-7]. In this study, we found that alkaline phosphatase was positive 2 weeks after induction in children, osteocalcin gene was obvious 3 weeks later, and calcium node appeared after Von Kossa staining 4 weeks later. It was shown that BMSCs of children have a high potential of directional differentiation into osteoblasts, and can be considered as seed cells in bone tissue engineering.
PPARγ-2 is the key factor of BMSCs differentiating into adipocytes. Lipid droplet is the characteristic of adipocytes. Thus, both of them can be regarded as the marker of BMSCs differentiating into adipocytes[8]. In our study, dexamethasone, insulin, 3-Isobutyl-1-methylxanthine and indomethacin were used to induce BMSCs. Lipid droplet gradually appeared in BMSCs, became larger and changed into fat vacuole. Simultaneously, PPARγ-2 highly expressed in BMSCs by reverse transcription polymerase chain reaction. Therefore, the differentiation of BMSCs into adipocytes was successful. The mechanisms of BMSCs differentiating into adipocytes are unclear. Several inductors possibly induce the generation of genes, which were related to the differentiation of BMSCs into adipocytes[9]. Hung SC et al[10] found that 88 genes and 31 genes respectively amplified at least 5 times and twice in the differentiation of BMSCs into adipocytes by microarray technology and reverse transcription polymerase chain reaction. PPARγ-2, C/EBP and 1 (ADD1-SRFT3P) played important roles in the differentiation of BMSCs into adipocytes[11]. Dexamethasone induces the generation of C/EBPδ by stimulating glucocorticoid receptor, and dexamethasone decreases the expression of Preadipocyte factor-1 (pref-1). 3-isobutyl-1-methylxanthine is a specific inhibitor of phosphodiesterase, can elevate the cyclic adenosine monophosphate level by preventing the degradation of cyclic adenosine monophosphate. Cyclic adenosine monophosphate that is a kind of important adipocyte inductor[12] can adjust the expression of C/EBPα and C/EBPβ by activating cyclic adenosine monophosphate response element binding protein, and then accelerate the generation of adipocytes. Insulin combined with insulin-like growth factor-I can adjust the phosphorylation and transcription activity of cyclic adenosine monophosphate response element binding protein by activating Akt, Ras, ERK1/ERK2 and reducing PP2A activity. Cyclic adenosine monophosphate response element binding protein after activation can accelerate the C/EBP level and increase the PPARγ-2 level. Some scholars[13] found that some BMSCs could differentiate into adipocytes, but others could differentiate into osteoblasts under a certain condition; The more the adipocytes, the less the osteoblasts had, and the less the adipocytes, the more the osteoblasts had; The amount of differentiated cells was mainly associated with the concentration of dexamethasone.
Latest studies showed that BMSCs not only could differentiate into osteoblasts, chondroblasts and lipoblasts, but also ectoderm cells under a certain condition. According to the study by Woodbury et al [14], we used β-mercaptoethanol, dimethyl sulphoxide and butylated hydroxyanisole for induction; neuron specific enolase and neurofilament-200 protein were positive, and nissl body appeared after toluidine blue staining. It was shown that BMSCs had successfully differentiated into neuron-like cells and had the antigen property. Mitogen-activated protein kinase participated in the differentiation into neural cells. Mitogen-activated protein kinase is the specific serine/threonine protein kinase. By causing threonine and tyrosine residue, ecto-stimulator can activate mitogen-activated protein kinase, which can activate transcription factors and adjust genes by activating the substrate molecule. Some cells died and disintegrated during the differentiation of BMSCs into neuron-like cells with the same method. It might be associated with the apoptosis caused by β-mercaptoethanol and butylated hydroxyanisole. Chen et al[15] also usedβ-mercaptoethanol for the differentiation of BMSCs into neural cells. However, the cell survival rate was higher and the survival time was longer after adding basic fibroblast growth factor and brain-derived neurotrophic factor before and after induction.
BMSCs are characterized by easily collecting materials, in vitro isolation, culture, amplification, and no immunological rejection after autotransfusion, high gene transfection efficiency and stability after transfection. Thus, BMSCs are considered as important seed cells in tissue engineering and gene engineering. Because of the specificity of children individual and disease entities, some scholars[8,16-17] found that the proliferation and differentiation of BMSCs were high after a primary comparison on biological characteristic of BMSCs from children of different ages. These brought great hopes for the study of children cell tissue engineering, and might offer a new therapeutic method for children congenital diseases such as inborn error of metabolism[18], maldevelopment of tibia,
Perthe's disease, spinal cord tethered cord syndrome, congenital cardiopathy, giant colon and so on. Scholars had obtained some results with the application of BMSCs in neural cell transplantation for experimental giant colon[19-20].

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小儿骨髓间充质干细胞生物学特性及 多向分化潜能☆

邵景范1，黎润光1，魏明发1，杨小进1，柴成伟1，覃宇冰1，康慧聪2，赵东明3，杨 勇3
华中科技大学同济医学院附属同济医院，1小儿外科，2神经内科， 3骨科，湖北省武汉市 430030
邵景范☆，男，1965生，汉族，浙江省慈溪县人，副教授，副主任医师， 1997年德国乌尔姆大学毕业，医学博士，主要从事小儿骨科以及细胞工程研究。
摘要
背景：国内报道人骨髓间充质干细胞多取材于成人细胞，小儿骨髓间充质干细胞的研究较少。
目的：拟分离培养小儿的骨髓间充质干细胞，观察其生物学特性以及向成骨、成脂肪、成神经分化的潜能。
设计：观察对比实验。
单位：华中科技大学同济医学院。
材料：实验于2006-03/09在武汉同济医院骨科实验室完成，小儿骨髓间充质干细胞取自5~8岁因髋关节发育不良行骨盆截骨术的患儿，男1例，女2例，实验经过医院伦理委员会批准许可，并征得所有患儿家属知情同意。地塞米松、维生素C、β-甘油磷酸钠、甲基异丁酸黄嘌呤、胰岛素、吲哚醚辛、羟基丁酸苯甲醚均为Sigma 公司产品，二甲基亚砜为Amersco公司产品。
方法：经Percoll梯度分离接种获得小儿骨髓间充质细胞，倒置相差显微镜下观察细胞形态、排列分布情况。分别采用成骨细胞诱导剂（β-甘油磷酸钠，地塞米松，维生素C）、成脂肪细胞诱导液（地塞米松，甲基异丁酸黄嘌呤, 牛胰岛素, 吲哚美辛）及二甲基亚砜和羟基丁酸苯甲醚无血清培养基诱导剂干预细胞向成骨、脂肪、神经细胞分化，经免疫组织化学染色、PCR、免疫细胞染色方法检测成骨标志物（碱性磷酸酶、骨钙素mRNA、钙结节）、脂肪标志物（脂滴、PPARγ-2mRNA）、以及类神经标志物（尼克氏体、神经烯醇化酶、神经丝蛋白）。
主要观察指标：①小儿骨髓间充质细胞形态以及增殖情况。②成骨、脂肪及类神经标志物检测结果。
结果：①小儿骨髓间充质细胞贴壁容易，细胞细长梭形，增殖能力强，融合后呈旋涡状分布。②骨髓间充质细胞分别经各诱导剂诱导后，细胞形态均发生相应的变化，用化学染色、PCR、免疫细胞染色等方法检测成骨标志物、脂肪标志物及类神经标志物有明显阳性表达。
结论：小儿骨髓间充质细胞生长具有稳定增殖、传代的能力，具有成骨、成脂肪、成神经等多方向的定向分化潜能。
关键词：小儿；骨髓间充质干细胞；增殖；多向分化
中图分类号: R394.2 文献标识码: A 文章编号: 1673-8225(2008)12-02369-05
邵景范，黎润光，魏明发，杨小进，柴成伟，覃宇冰，康慧聪，赵东明，杨勇.小儿骨髓间充质干细胞生物学特性及多向分化潜能[J].中国组织工程研究与临床康复，2008，12(12):2369-2373
[www.zglckf.com/zglckf/ejournal/upfiles/08-12/12k-2369(ps).pdf]
(Edited by Olafur E. Sigurjonsson/Qiu Y/Wang L)