Peng Hsiu-Huei, Wang Tzu-Hao, Chao An-Shine, Chang Shuenn-Dyh
Department of Obstetrics and Gynecology, Chang Gung Memorial Hospital, Taipei, Chang Gung University College of Medicine, Taoyuan, Taiwan.
Chang Gung Med J. 2007 Sep-Oct;30(5):402-7.
The aims of this study were to evaluate the efficacy of current techniques for isolating mesenchymal stem cells (MSCs) from amniotic fluid obtained by second-trimester amniocentesis as well as to determine their differentiation potential.
We collected 50 samples of amniotic fluid by second-trimester amniocentesis. To obtain MSCs from amniotic fluid, the fluid was cultured using the two-stage culture protocol described in previous literature. Reverse transcription-polymerase chain reaction (RT-PCR) of a stem cell-specific transcription factor, octamer-binding protein 4 (Oct-4), was used to identify the characteristics of the MSCs cultured from amniotic fluid. Osteogeneic differentiation of these MSCs was confirmed by the presence of osteocalcin (a mineral-binding protein uniquely synthesized by osteoblasts) using RT-PCR and Von Kossa staining. Adipogenic differentiation of these MSCs was displayed by RT-PCR of adipocyte lipid-binding protein (a lipid-binding protein specifically in adipocytes) and Oil Red O staining.
Amniotic fluid-derived MSCs were successfully isolated and cultured from six samples. These cells could express the pluripotent stem cell-specific transcription factor Oct-4 as confirmed by RT-PCR. Under specific culture conditions, amniotic fluid-derived MSCs could be successfully induced to differentiate into adipocytes and osteocytes, based on product analysis by RT-PCR and specific staining.
Based on our experiment, we estimate the efficacy of isolating mesenchymal stem cells from second-trimester amniotic fluid obtained by amniocentesis to be about 12%. Human MSCs from second-trimester amniotic fluid had the ability to differentiate in vitro into adipocytes and osteocytes under specific culture conditions. The multilineage differentiation potential of these amniotic fluid-derived MSCs may be applicable to cell transplantation and regenerative medicine.
本研究旨在评估当前从孕中期羊膜腔穿刺获得的羊水分离间充质干细胞（MSCs）技术的有效性，并确定其分化潜能。
我们通过孕中期羊膜腔穿刺收集了50份羊水样本。为了从羊水中获取MSCs，采用先前文献中描述的两阶段培养方案对羊水进行培养。使用干细胞特异性转录因子八聚体结合蛋白4（Oct-4）的逆转录-聚合酶链反应（RT-PCR）来鉴定从羊水中培养的MSCs的特征。通过RT-PCR检测骨钙素（一种由成骨细胞独特合成的矿物质结合蛋白）的存在以及采用冯科萨染色法，证实这些MSCs的成骨分化。通过脂肪细胞脂质结合蛋白（一种专门存在于脂肪细胞中的脂质结合蛋白）的RT-PCR以及油红O染色，展示这些MSCs的成脂分化。
成功从6份样本中分离并培养出羊水来源的MSCs。经RT-PCR证实，这些细胞能够表达多能干细胞特异性转录因子Oct-4。在特定培养条件下，基于RT-PCR产物分析和特异性染色，羊水来源的MSCs能够成功诱导分化为脂肪细胞和骨细胞。
基于我们的实验，我们估计通过羊膜腔穿刺从孕中期羊水中分离间充质干细胞的成功率约为12%。孕中期羊水来源的人MSCs在特定培养条件下具有体外分化为脂肪细胞和骨细胞的能力。这些羊水来源的MSCs的多系分化潜能可能适用于细胞移植和再生医学。
