Osteogenic and chondrogenic differentiation by adipose-derived stem cells harvested from GFP transgenic mice
Osteogenic and chondrogenic differentiation by adipose-derived stem cells harvested from GFP transgenic mice
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DOI：
10.1016/j.bbrc.2003.12.017
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发表时间：
2004-01-23
影响因子：
3.1
通讯作者：
Hyakusoku, H
中科院分区：
文献类型：
--
作者：
Ogawa, R;Mizuno, H;Hyakusoku, H
最近的研究表明，人类脂肪组织含有类似于骨髓来源的干细胞的多能干细胞。利用均匀标记的细胞从绿色荧光蛋白（GFP）转基因小鼠，我们以前已经证明，骨髓来源的基质细胞（BSC）分化成各种细胞谱系在体外和体内。在本研究中，我们扩展了这种方法来表征脂肪组织来源的基质细胞，有时被称为处理过的脂肪抽吸物（PLA）细胞。脂肪来源的基质细胞（ASC）分离自腹股沟脂肪垫的GFP转基因小鼠后，广泛的洗涤与磷酸盐缓冲盐水和处理与胶原酶。在对照培养基中原代培养后（Dulbecco改良Eagle培养基+10%胎牛血清）并扩增至两代，将细胞在成骨培养基中孵育（Dulbecco改良Eagle培养基+10%胎牛血清+地塞米松+抗坏血酸-2-磷酸盐+ β-甘油磷酸盐）或软骨形成培养基（Dulbecco改良Eagle培养基+1%胎牛血清+胰岛素+抗坏血酸-2-磷酸+转化生长因子-β 1）培养2-4周，分别诱导成骨和软骨形成。通过von Kossa和碱性磷酸酶染色评估成骨分化，而通过阿尔新蓝染色评估软骨分化。通过RT-PCR证实骨细胞特异性骨桥蛋白、骨钙素和碱性磷酸酶以及软骨细胞特异性聚集蛋白聚糖和II/X型胶原的表达。在成骨培养基中培养的ASCs对von Kossa和碱性磷酸酶染色呈阳性。除骨钙素外，还检测了骨细胞特异性基因的表达。与软骨形成培养基孵育诱导阿辛蓝阳性细胞和聚集蛋白聚糖和II/X型胶原基因的表达。在对照培养基中孵育的细胞中未观察到骨软骨形成分化。来自GFP转基因小鼠的ASCs在体外具有成骨和成软骨潜能。由于这种细胞群可以很容易地通过荧光显微镜识别，它可能是一个理想的来源，为进一步的干细胞生物学和组织工程实验的ASCs。(C)2003年爱思唯尔公司All rights reserved.
Recent studies suggest that human adipose tissue contains pluripotent stem cells similar to bone marrow-derived stem cells. Taking advantage of homogeneously marked cells from green fluorescent protein (GFP) transgenic mice, we have previously demonstrated that bone marrow-derived stromal cells (BSCs) differentiate into a variety of cell lineages both in vitro and in vivo. In the present study, we extend this approach to characterize adipose tissue-derived stromal cells, sometimes called processed lipoaspirate (PLA) cells. Adipose-derived stromal cells (ASCs) were isolated from inguinal fat pads of GFP transgenic mice after extensive washing with phosphate-buffered saline and treatment with collagenase. After primary culture in a control medium (Dulbecco's modified Eagle's medium + 10% fetal bovine serum) and expansion to two passages, the cells were incubated in either an osteogenic medium (Dulbecco's modified Eagle's medium + 10% fetal bovine serum + dexamethasone + ascorbate-2-phosphate + beta-glycerophosphate) or a chondrogenic medium (Dulbecco's modified Eagle's medium+ 1% fetal bovine serum+ insulin+ ascorbate-2-phosphate+ transforming growth factor-beta1) for 2-4 weeks to induce osteogenesis and chondrogenesis, respectively. Osteogenic differentiation was assessed by von Kossa and alkaline phosphatase staining, while chondrogenic differentiation was assessed by Alcian blue staining. Expression of osteocyte specific osteopontin, osteocalcin, and alkaline phosphatase, and chondrocyte specific aggrecan and type II/X collagen was confirmed by RT-PCR. ASCs incubated in the osteogenic medium were stained positively for von Kossa and alkaline phosphatase staining. Expression of osteocyte specific genes, except osteocalcin, was also detected. Incubation with chondrogenic medium induced Alcian blue positive cells and expression of aggrecan and type II/X collagen genes. No osteochondrogenic differentiation was observed in cells incubated in the control medium. ASCs from GFP transgenic mice have both osteogenic and chondrogenic potential in vitro. Since this cell population can be easily identified through fluorescence microscopy, it may be an ideal source of ASCs for further experiments on stem cell biology and tissue engineering. (C) 2003 Elsevier Inc. All rights reserved.