Bone marrow mesenchymal stem cells from Sprague-Dawley rats: aging inhibits cell proliferation and differentiation

doi:10.3969/j.issn.2095-4344.2014.32.005

Abstract

Abstract:

BACKGROUND: Bone marrow mesenchymal stem cells are ideal as tissue engineering seed cells, but the proliferation and differentiation of mesenchymal stem cells in vitro is very different at different ages. Moreover, there are few reports on the association between age and the number of bone marrow mesenchymal stem cells.
OBJECTIVE: To observe the difference in differentiation ability of bone marrow mesenchymal stem cells from rats at different ages.
METHODS: We isolated, purified and amplified the mesenchymal stem cells from rat bone marrow in vitro by the whole bone marrow adherent culture; observed the morphological characteristics of mesenchymal stem cells under an inverted phase contrast microscope; detected cell surface markers by flow cytometer. Then, mesenchymal stem cells were induced in vitro into osteoblasts and chondroblasts and verified. Passage 3 cells from rats at ages of 2, 4, 6, 8, 12 weeks and 10, 12 months were subjected to osteogenic induction at weeks 1, 2, 3. ELISA was used to determine osteocalcin content.
RESULTS AND CONCLUSION: Bone marrow mesenchymal stem cells cultured in vitro were adherent and exhibited a fibroblast-like spindle shape. In vitro, cells proliferated quickly to form colonies. Flow cytometry showed that the cells were positive for CD29, CD90, but negative for CD45, and partially expressed CD44. After osteogenic induction, cells were positive for alkaline phosphatase staining and alizarin red staining; after chondrogenic induction, cells were positive for alcian blue staining. Mesenchymal stem cells could be isolated and cultured by the method of bone marrow adherent culture in vitro. However, bone marrow mesenchymal stem cells from rats at different ages exhibit decreased proliferation and differentiation abilities with the increase of age through determination of osteocalcin content.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

全文链接：

Key words: bone marrow, mesenchymal stem cells, rats, Sprague-Dawley, osteocalcin

CLC Number:

R394.2

Cui Xin-tao, Tao Shu-qing. Bone marrow mesenchymal stem cells from Sprague-Dawley rats: aging inhibits cell proliferation and differentiation[J]. Chinese Journal of Tissue Engineering Research, 2014, 18(32): 5108-5113.

Figures/Tables 2

2.1 骨髓间充质干细胞的形态学观察 2.1.1 原代培养原代培养细胞刚接种时，培养瓶中有大量悬浮细胞，未贴壁的细胞逐渐坏死破碎，在换液过程中逐步被清除。原代培养12 h后细胞开始贴壁。24 h后部分细胞开始贴壁伸展变形，刚贴壁的细胞单个排列，呈圆形或多角形，胞体透亮折光性强(图1A)。72 h后呈纺锤形或梭形，附壁铺伸细胞数量增多，呈多个散在分布的细胞集落(图1B)。6 d后见贴壁生长的细胞数量明显增多，细胞形态出现较大变化，可见成纤维样细胞散布于培养瓶底，并有多个集落形成，细胞围绕中心呈漩涡状排列(图1C)。9 d后集落逐渐增多，此后逐渐增大，并融合为单层(图1D)。当培养至12 d时，细胞已大部分融合，长满瓶底，细胞呈长梭形(成纤维样外观)，有些呈交叉重叠生长(图1E)。 2.1.2 传代培养原代细胞培养12-14 d后，消化传代，传代细胞贴壁较快，刚传代的细胞悬浮于培养液中，圆形，核透亮，“满天星”样分布(图2A)。2 h后细胞开始贴壁，细胞形态由圆形向梭形转化。4 h后可见明显的贴壁细胞散在分布。24 h见贴壁细胞呈单层生长，伸展为短梭形、长梭形。原代培养局部区域存在少许小而圆的细胞，传代后消失(图2B)。3-5 d细胞长满培养瓶底，形态为成纤维细胞样，排列整齐有序(图2C)。第3代以后，细胞形态趋于一致。传代细胞可连续传7-10代，细胞形状基本相同。但随着传代次数增多，细胞逐渐老化，表现为：细胞增殖速度明显减慢，胞浆中出现空泡及颗粒样物质，细胞碎片增多，细胞形态变为扁平，失去贴壁能力，从培养瓶底脱落。 2.2 骨髓间充质干细胞的表面抗原鉴定骨髓间充质干细胞的表面抗原鉴定结果CD29、CD90表达阳性，CD44部分表达，CD45表达阴性(图3)。 2.3 诱导分化能力 2.3.1 成骨诱导分化诱导约7 d时细胞呈多角形，胞质内细胞颗粒增多；10 d时胞质内充满颗粒，细胞呈集落样生长，细胞间可见钙质沉积；15 d时细胞结节中心的细胞逐渐融合失去细胞结构，钙结节形成明显，19 d时经茜素红染色呈红色结节(图4A)。 2.3.2 成软骨诱导分化诱导过程中，细胞团直径会有所增大，表面会变得光滑并呈胶质状。诱导19 d时经阿利辛蓝染色呈淡蓝色(图4B)。 2.3.3 碱性磷酸酶染色诱导1，3，7，10 d后染色呈阴性，第16天后染色呈阳性(图4C，D)。 2.4 骨钙素定量检测从表1中可以看出不同鼠龄骨髓间充质干细胞的骨钙素水平之间存在差异，2周龄，4周龄组的骨钙素水平明显高于其他组。2，4周龄组与12周龄组之间比较差异无显著性意义(P > 0.05)，2，4周龄组与10月龄组之间比较差异有显著性意义(P < 0.05)，2，4周龄组与12月龄组之间比较差异非常显著性意义(P < 0.01)。"

References

[1] Friedenstein AJ, Deriglasova UF, Kulagina NN,et al. Precursors for fibroblasts in different populations of hematopoietic cells as detected by the in vitro colony assay method.Exp Hematol. 1974;2(2):83-92.
[2] Vidal MA, Kilroy GE, Lopez MJ, et al. Characterization of equine adipose tissue-derived stromal cells: adipogenic and osteogenic capacity and comparison with bone marrow-derived mesenchymal stromal cells.Vet Surg. 2007; 36(7):613-622.
[3] Kobayashi N, Yasu T, Ueba H,et al. Mechanical stress promotes the expression of smooth muscle-like properties in marrow stromal cells.Exp Hematol. 2004;32(12):1238-1245.
[4] Zhang Y, Zhao L, Tang H. Isolation and cultivation of mouse bone marrow mesenchymal stem cells.Sheng Wu Yi Xue Gong Cheng Xue Za Zhi. 2004;21(5):746-748, 765.
[5] Nadri S, Soleimani M, Hosseni RH, et al. An efficient method for isolation of murine bone marrow mesenchymal stem cells. Int J Dev Biol. 2007;51(8):723-729.
[6] Phinney DG, Kopen G, Isaacson RL,et al. Plastic adherent stromal cells from the bone marrow of commonly used strains of inbred mice: variations in yield, growth, and differentiation.J Cell Biochem. 1999;72(4):570-585.
[7] Conget PA, Minguell JJ. Phenotypical and functional properties of human bone marrow mesenchymal progenitor cells.J Cell Physiol. 1999;181(1):67-73.
[8] Kim HJ, Zhao H, Kitaura H, et al. Glucocorticoids suppress bone formation via the osteoclast.J Clin Invest. 2006;116(8): 2152-2160.
[9] Jäger M, Fischer J, Dohrn W,et al. Dexamethasone modulates BMP-2 effects on mesenchymal stem cells in vitro.J Orthop Res. 2008;26(11):1440-1448.
[10] Gigante A, Torcianti M, Boldrini E,et al. Vitamin K and D association stimulates in vitro osteoblast differentiation of fracture site derived human mesenchymal stem cells.J Biol Regul Homeost Agents. 2008;22(1):35-44.
[11] Meng E, Guo Z, Wang H,et al. High mobility group box 1 protein inhibits the proliferation of human mesenchymal stem cells and promotes their migration and differentiation along osteoblastic pathway.Stem Cells Dev. 2008;17(4):805-813.
[12] Xu J, Wang W, Ludeman M, et al. Chondrogenic differentiation of human mesenchymal stem cells in three-dimensional alginate gels.Tissue Eng Part A. 2008; 14(5):667-680.
[13] Mackay AM, Beck SC, Murphy JM,et al. Chondrogenic differentiation of cultured human mesenchymal stem cells from marrow.Tissue Eng. 1998;4(4):415-428.
[14] Pelaez D, Huang CY, Cheung HS.Cyclic compression maintains viability and induces chondrogenesis of human mesenchymal stem cells in fibrin gel scaffolds.Stem Cells Dev. 2009;18(1):93-102.
[15] 郝瑞锋,申长虹.骨髓间充质干细胞向成骨细胞诱导的研究现状[J].天津医科大学学报,2007,13(1):129-131.
[16] Vidal MA, Kilroy GE, Lopez MJ,et al. Characterization of equine adipose tissue-derived stromal cells: adipogenic and osteogenic capacity and comparison with bone marrow-derived mesenchymal stromal cells.Vet Surg. 2007; 36(7):613-622.