Biological characteristics and differentiation potential of bone marrow embryonic-like stem cells in rats

doi:10.3969/j.issn.2095-4344.2017.01.008

Abstract

Abstract:

BACKGROUND: Rat bone marrow embryonic-like stem cells are found to express embryonic stem cell markers, differentiate into cells derived from three germ layers, and improve cardiac function and remodel the ventricle after implantation. However, the cell biological characteristics and differentiation potential are still unclear.
OBJECTIVE: To study the biological properties and differentiation potential of embryonic-like stem cells from rat bone marrow.
METHODS: Bone marrow mononuclear cells from 20 Sprague-Dawley were labeled with stage-specific embryonic antigen 1 (SSAE-1), and bone marrow embryonic stem cells were sorted by flow cytometry. Morphology and structure of the sorted cells were observed under scanning electron microscope. Expression of transcription factor (Oct4, Nanog, Sox2) in bone marrow embryonic-like stem cells was detected by immunofluorescence staining. Bone marrow embryonic-like stem cells were cultured and amplified in the feeder layer of fibroblasts to induce myocardium and endothelial cells, and their specific proteins expression was determined.
RESULTS AND CONCLUSION: SSEA-1 labeled rat bone marrow embryonic-like stem cells were gated based on a diameter of 2-6 μm, and these cells were fusiform, polygonal or circular, with a small size. Under the transmission electron microscope, the cell nucleus was enlarged with less cytoplasm containing a small amount of organelles. Multipotential antigen markers could be detected under the immunocytochemical staining of passage 2 rat bone marrow embryonic-like stem cells. With the increase of the number of passages, the cell proliferation ability exhibited a descending trend. One week after myocardial differentiation of rat bone marrow embryonic-like stem cells, myocardial cells were in the parallel arrangement. Two weeks after myocardial induction, the cell morphology changed significantly, and interconnected cells were observed. Immunofluorescence staining showed that cTnT and Cx43 protein were expressed in the myocardium. One week after endothelial differentiation, the endothelial cells were polygonal and spindle-shaped. Two weeks after endothelial differentiation, immunocytochemical staining showed that VWF and CD31 were expressed in the interconnected endothelial cells. To conclude, these findings indicate that rat bone marrow embryonic-like stem cells have similar biological characteristics as bone marrow mesenchymal stem cells and can express stem cell transcription factors. These cells are a likely to differentiate into myocardial and endothelial cells under certain conditions and have a high clinical value.

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

Key words: Stem Cells, Cardiovascular Diseases, Heart Diseases, Embryonic Stem Cells

CLC Number:

R394.2

Wang Zheng, Lin Kai. Biological characteristics and differentiation potential of bone marrow embryonic-like stem cells in rats[J]. Chinese Journal of Tissue Engineering Research, 2017, 21(1): 43-48.

Figures/Tables 1

2.1 实验动物数量分析实验选用SD大鼠20只，中途无脱落，全部进行结果分析。 2.2 大鼠骨髓胚胎样干细胞分离、鉴定采用SSEA-1标记大鼠骨髓胚胎样干细胞，对直径为2-6 μm细胞设门，倒置显微镜下大鼠骨髓胚胎样干细胞体积较小，形态相对均以。同时，大鼠骨髓胚胎样干细胞呈现纺锤形，多边形以及圆形多细胞形态，见图1。 2.3 大鼠骨髓胚胎样干细胞扩增结果大鼠骨髓胚胎样干细胞体积较小，直径为4.0-5.0 μm，透射电镜下可见细胞核开始增大，细胞质较少，含有少量的细胞器。在无饲养层的培养条件下大鼠骨髓胚胎样干细胞增殖不明显。将大鼠骨髓胚胎样干细胞接种在纤维细胞饲养层上1 d细胞开始黏附分布。培养7 d，细胞增殖形成细胞簇，细胞较小，见图2。在扫描电镜下，大鼠骨髓胚胎样干细胞表面含有较多的微绒毛，并且大鼠骨髓胚胎样干细胞开始向外扩展，见图3。 2.4 免疫荧光染色结果采用无血清培养基扩增的第2代大鼠骨髓胚胎样干细胞，结果显示：免疫荧光染色下能测定到多潜能抗原标志，如：Oct-4、Nanog-3和Sox-2，但是表达相对微弱，见图4。 2.5 大鼠骨髓胚胎样干细胞生长曲线经过酶标仪测定A值并进行比较，结果显示：大鼠骨髓胚胎样干细胞在传代生长过程中随着传代次数的不断增加，细胞增殖能力出现下降趋势，且每次传代经历两三天，经过2-5 d后经历对数增殖期而进入平台期，见表1。从图5看出：大鼠骨髓胚胎样干细胞不同传代数的增加，细胞总体呈现上升趋势，但是随着传代次数增加，细胞生长能力变低。 2.6 大鼠骨髓胚胎样干细胞分化潜力实验大鼠骨髓胚胎干细胞分化1周后心肌细胞平行排列，诱导2周后细胞形态发生明显的变化，细胞相互连接。免疫细胞化学染色可见心肌特异蛋白cTnT以及Cx43表达，见图6。大鼠骨髓胚胎样干细胞分化1周后内皮细胞呈现多角形、梭形。诱导2周后细胞相互连接，免疫细胞化学染色可见内皮细胞特异蛋白VWF以及CD31表达，见图7。"

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