Distinct gene expression patterns of ‘multipotent' versus ‘unipotent' single colony-derived strains (SCDSs) of human bone marrow stromal cells (BMSCs)

Dyskeratosis congenita (DC) is an inherited multi-system disorder, characterized by oral leukoplakia, nail dystrophy, and abnormal skin pigmentation, as well as high rates of bone marrow failure, solid tumors, and other medical problems such as osteopenia. DC and telomere biology disorders (collectively referred to as TBD here) are caused by germline mutations in telomere biology genes leading to very short telomeres and limited proliferative potential of hematopoietic stem cells. We found that skeletal stem cells (SSCs) within the bone marrow stromal cell population (BMSCs, also known as bone marrow-derived mesenchymal stem cells), may contribute to the hematological phenotype. At least 5x105 cells from each SCDS were homogenized in Trizol (Invitrogen) for RNA extraction. RNA was purified by using a combination of chloroform phase separation and RNeasy Mini Kits (Qiagen) according to the manufacturer's protocol. RNA was quantified and 5ug was processed for microarray analysis (LMT, NCI, Frederick, MD). RNA was reverse transcribed to form cDNA, and hybridized to Affymetrix GeneChip Human Genome U133 Plus 2.0 arrays. Three independent replicates for each of the experimental conditions were carried out and analyzed to control for intra-sample variation.

先天性角化不良症（Dyskeratosis congenita, DC）是一种遗传性多系统疾病，以口腔白斑、指甲营养不良、皮肤色素异常为典型特征，且伴随骨髓衰竭、实体瘤及骨质减少等多种病症的高发生率。DC与端粒生物学障碍（telomere biology disorders, 下文统称TBD）均由端粒相关基因的生殖系突变引发，可导致端粒显著缩短及造血干细胞增殖潜能受限。本研究发现，骨髓基质细胞群（BMSCs，亦称骨髓来源间充质干细胞）内的骨骼干细胞（SSCs）或参与血液系统表型的发生。我们从每份SCDS中获取至少5×10^5个细胞，使用TRIzol试剂（Invitrogen公司）进行匀浆以提取RNA。随后采用氯仿相分离法与RNeasy迷你试剂盒（Qiagen公司），严格遵循制造商实验方案纯化RNA。对纯化后的RNA进行定量，取5μg用于微阵列分析（由美国马里兰州弗雷德里克市国家癌症研究所LMT实验室完成）。将RNA反转录为互补脱氧核糖核酸（cDNA）后，与Affymetrix GeneChip人类基因组U133 Plus 2.0芯片进行杂交。每个实验条件均设置3次独立重复实验并开展分析，以控制样本内变异带来的干扰。