Co-culture of adipose-derived stem cells and osteoblasts under different conditions

doi:10.3969/j.issn.2095-4344.2014.37.018

Abstract

Abstract:

BACKGROUND: After co-culture with osteoblasts, bone marrow stem cells can be induced to differentiate into osteoblasts. Whether adipose-derived stem cells co-cultured with osteoblasts can differentiate into osteoblasts or not?
OBJECTIVE: To observe whether adipose-derived stem cells co-cultured with osteoblasts can differentiate into osteoblasts.
METHODS: Adipose-derived stem cells and osteoblasts were isolated from New Zealand white rabbits. Then, passage 3 adipose-derived stem cells were co-cultured with passage 2 osteoblasts in 10% or 5% fetal bovine serum for 14 days.
RESULTS AND CONCLUSION: After 7 days of co-culture, some adipose-derived stem cells became round in the two groups. After 14 days of co-culture, adipose-derived stem cells highly differentiated and differentiated cells were similar to mature osteoblasts that were positive for alkaline phosphatase staining and alizarin red staining. The mRNA expression of type I collagen and osteocalcin increased in both two group, especially in the 10% fetal bovine serum group. These findings indicate that adipose-derived stem cells co-cultured with osteoblasts can differentiate into osteoblasts induced by high-concentration serum culture.

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: adipocytes, osteoblasts, cell differentiation, cell culture techniques

CLC Number:

R394.2

Zhang Yang, Liu Da-cheng, Yang Xiao-ning. Co-culture of adipose-derived stem cells and osteoblasts under different conditions[J]. Chinese Journal of Tissue Engineering Research, 2014, 18(37): 6003-6007.

Figures/Tables 2

2.1 分离培养的脂肪干细胞首次分离的细胞形态呈小圆形，培养24 h后开始贴壁，未贴壁细胞为椭圆形红细胞，大部分贴壁细胞呈梭形、多角形，细胞首次换液后，未贴壁细胞被去除。随培养时间的延长，细胞数量增多，形态为典型的长梭形，呈集落式、团簇状生长。细胞融合达80%-90%。3代细胞呈“漩涡状”生长，并能长期稳定培养(图1A，B)。免疫荧光染色法检测显示，细胞表面抗原CD44呈阳性表明这种细胞为间充质干细胞；而CD45呈阴性证明所检测细胞并非来源于血液循环中的干细胞(图1C，D)。综上可以初步鉴定所分离的细胞为脂肪干细胞。 2.2 共培养后的脂肪干细胞的形态学变化细胞共培养 7 d后，部分脂肪干细胞变成多角形、圆形及不规则形状，增殖速度减慢，其中以10%胎牛血清组改变最为明显(图2A，B)。共培养14 d后，10%胎牛血清共培养组脂肪干细胞形态学表现与成骨细胞最为接近，5%胎牛血清共培养组也有成骨细胞形态，但是细胞有老化表现(图2C，D)。 2.3 成骨细胞分化后脂肪干细胞的碱性磷酸酶染色及茜素红染色鉴定成骨诱导14 d时，2组细胞碱性磷酸酶染色均为阳性，倒置相差显微镜下可见大量细胞核为深蓝色，胞浆为蓝黑色，但10%胎牛血清共培养组的阳性细胞数量明显较5%胎牛血清共培养组多。成骨诱导14 d时，2组细胞茜素红染色均呈阳性，镜下可见10%胎牛血清共培养组细胞中钙结节明显较5%胎牛血清共培养组多且体积大(图3，表1)。 2.4 脂肪干细胞成骨分化后I型胶原和骨钙素mRNA的表达反转录PCR检测显示，成骨诱导7，14 d后，2组细胞均检测到Ⅰ型胶原和骨钙素mRNA的表达，且从灰度上观察两组表达均随时间越来越强，从组别来看实验组改变更为明显，从表达产物上观察Ⅰ型胶原mRNA的表达更丰富，从同一时间点观察实验组Ⅰ型胶原和骨钙素mRNA的表达均强于对照组(图4)。"

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