Ogawa Rei, Mizuno Hiroshi, Watanabe Atsushi, Migita Makoto, Shimada Takashi, Hyakusoku Hiko
Department of Plastic and Reconstructive Surgery, Nippon Medical School, 1-1-5 Sendagi, Bunkyo-ku, Toky 113-8603, Japan.
Biochem Biophys Res Commun. 2004 Jan 23;313(4):871-7. doi: 10.1016/j.bbrc.2003.12.017.
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.
最近的研究表明，人体脂肪组织含有与骨髓来源干细胞相似的多能干细胞。利用来自绿色荧光蛋白（GFP）转基因小鼠的均匀标记细胞，我们之前已经证明骨髓来源的基质细胞（BSCs）在体外和体内均可分化为多种细胞谱系。在本研究中，我们将这种方法扩展至对脂肪组织来源的基质细胞（有时称为处理过的脂肪抽吸物（PLA）细胞）进行表征。脂肪来源的基质细胞（ASCs）从GFP转基因小鼠的腹股沟脂肪垫中分离出来，先用磷酸盐缓冲盐水充分洗涤，再用胶原酶处理。在对照培养基（杜氏改良 Eagle 培养基 + 10%胎牛血清）中进行原代培养并传代至第二代后，将细胞分别在成骨培养基（杜氏改良 Eagle 培养基 + 10%胎牛血清 + 地塞米松 + 抗坏血酸 - 2 - 磷酸酯 + β - 甘油磷酸酯）或软骨形成培养基（杜氏改良 Eagle 培养基 + 1%胎牛血清 + 胰岛素 + 抗坏血酸 - 2 - 磷酸酯 + 转化生长因子 - β1）中孵育2 - 4周，以分别诱导成骨和软骨形成。通过冯·科萨染色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形成分化。通过逆转录 - 聚合酶链反应（RT - PCR）确认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型/X型胶原蛋白的表达。在成骨培养基中孵育的ASCs经冯·科萨染色和碱性磷酸酶染色呈阳性。除骨钙素外，还检测到骨细胞特异性基因的表达。用软骨形成培养基孵育可诱导阿尔新蓝阳性细胞以及聚集蛋白聚糖和II型/X型胶原蛋白基因的表达。在对照培养基中孵育的细胞未观察到成骨软骨分化。来自GFP转基因小鼠的ASCs在体外具有成骨和软骨形成潜能。由于通过荧光显微镜可以轻松识别这群细胞，它可能是用于干细胞生物学和组织工程进一步实验的理想ASCs来源。
