Warejcka D J, Harvey R, Taylor B J, Young H E, Lucas P A
Department of Surgery, Mercer University School of Medicine, Medical Center of Central Georgia, Macon 31208, USA.
J Surg Res. 1996 May;62(2):233-42. doi: 10.1006/jsre.1996.0201.
A population of stem cells has been isolated from embryonic avian and neonatal rat skeletal muscle. These cells differentiate into several mesodermal phenotypes in culture upon treatment with dexamethasone. This study reports the isolation of a similar population of stem cells from another mesodermal tissue, the heart. Hearts were excised from 3- to 5-day- old rats, minced, and treated with a collagenase-dispase solution. Single cells were collected by centrifugation, washed, and plated in dishes. The cells were grown to confluence, trypsinized, and frozen at -80 degrees C in 7.5% dimethylsulfoxide. After at least 24 hr, the cells were thawed and plated in 24-well plates and treated with media containing dexamethasone at concentrations of 10(-6)-10(-10) M for 4 weeks. Control cultures contained mononucleated cells with a stellate morphology. Treatment with dexamethasone resulted in the appearance of several mesodermal phenotypes. Bone and cartilage nodules were identified with von Kossa and Alcian blue staining respectively. Adipocytes were identified using Sudan black B stain. Smooth muscle cells were identified by an anti-smooth muscle alpha-actin antibody, and skeletal myotubes were stained with anti-myosin antibody. Large binuclear cells with obvious fibers were noted and stained with anti-desmin. These binuclear cells appeared in both the control and the dexamethasone-treated cultures and were tentatively identified as cardiomyocytes. These data strongly suggest the existence of a population of mesenchymal stem cells in neonatal rat heart.
已从胚胎期禽类和新生大鼠的骨骼肌中分离出一群干细胞。在用地塞米松处理后，这些细胞在培养中可分化为几种中胚层表型。本研究报告了从另一种中胚层组织——心脏中分离出类似的干细胞群体。从3至5日龄的大鼠中取出心脏，切碎，并用胶原酶- dispase溶液处理。通过离心收集单细胞，洗涤后接种于培养皿中。待细胞生长至汇合状态，用胰蛋白酶消化，然后在含有7.5%二甲基亚砜的条件下于-80℃冷冻保存。至少24小时后，将细胞解冻并接种于24孔板中，用含有浓度为10(-6)-10(-10)M地塞米松的培养基处理4周。对照培养物中含有具有星状形态的单核细胞。用地塞米松处理导致出现几种中胚层表型。分别用冯·科萨染色和阿尔辛蓝染色鉴定骨和软骨结节。用苏丹黑B染色鉴定脂肪细胞。用抗平滑肌α-肌动蛋白抗体鉴定平滑肌细胞，并用抗肌球蛋白抗体对骨骼肌肌管进行染色。观察到带有明显纤维的大型双核细胞，并用抗结蛋白进行染色。这些双核细胞在对照培养物和地塞米松处理的培养物中均出现，并初步鉴定为心肌细胞。这些数据有力地表明新生大鼠心脏中存在一群间充质干细胞。
