Johnstone B, Hering T M, Caplan A I, Goldberg V M, Yoo J U
Skeletal Research Center, Case Western Reserve University, Cleveland, Ohio 44106, USA.
Exp Cell Res. 1998 Jan 10;238(1):265-72. doi: 10.1006/excr.1997.3858.
A culture system that facilitates the chondrogenic differentiation of rabbit bone marrow-derived mesenchymal progenitor cells has been developed. Cells obtained in bone marrow aspirates were first isolated by monolayer culture and then transferred into tubes and allowed to form three-dimensional aggregates in a chemically defined medium. The inclusion of 10(-7) M dexamethasone in the medium induced chondrogenic differentiation of cells within the aggregate as evidenced by the appearance of toluidine blue metachromasia and the immunohistochemical detection of type II collagen as early as 7 days after beginning three-dimensional culture. After 21 days, the matrix of the entire aggregate contained type II collagen. By 14 days of culture, there was also evidence for type X collagen present in the matrix and the cells morphologically resembled hypertrophic chondrocytes. However, chondrogenic differentiation was achieved in only approximately 25% of the marrow cell preparations used. In contrast, with the addition of transforming growth factor-beta 1 (TGF-beta 1), chondrogenesis was induced in all marrow cell preparations, with or without the presence of 10(-7) M dexamethasone. The induction of chondrogenesis was accompanied by an increase in the alkaline phosphatase activity of the aggregated cells. The results of RT-PCR experiments indicated that both type IIA and IIB collagen mRNAs were detected by 7 days postaggregation as was mRNA for type X collagen. Conversely, the expression of the type I collagen mRNA was detected in the preaggregate cells but was no longer detectable at 7 days after aggregation. These results provide histological, immunohistochemical, and molecular evidence for the in vitro chondrogenic differentiation of adult mammalian progenitor cells derived from bone marrow.
已开发出一种促进兔骨髓间充质祖细胞软骨形成分化的培养系统。首先通过单层培养分离骨髓穿刺物中获得的细胞，然后将其转移至试管中，并在化学限定培养基中使其形成三维聚集体。培养基中加入10(-7) M地塞米松可诱导聚集体内细胞的软骨形成分化，早在三维培养开始7天后甲苯胺蓝异染性的出现以及II型胶原的免疫组织化学检测就证明了这一点。21天后，整个聚集体的基质中都含有II型胶原。培养14天时，基质中也有X型胶原存在的证据，且细胞在形态上类似于肥大软骨细胞。然而，仅约25%的所用骨髓细胞制剂实现了软骨形成分化。相比之下，添加转化生长因子-β1（TGF-β1）后，无论有无10(-7) M地塞米松，所有骨髓细胞制剂均诱导发生软骨形成。软骨形成的诱导伴随着聚集细胞碱性磷酸酶活性的增加。RT-PCR实验结果表明，聚集后7天可检测到IIA型和IIB型胶原mRNA以及X型胶原mRNA。相反，I型胶原mRNA在聚集前的细胞中可检测到，但聚集后7天不再能检测到。这些结果为成年哺乳动物骨髓来源祖细胞的体外软骨形成分化提供了组织学、免疫组织化学和分子证据。
