Guenther H L, Hofstetter W, Stutzer A, Mühlbauer R, Fleisch H
Department of Pathophysiology, University of Berne, Switzerland.
Endocrinology. 1989 Oct;125(4):2092-102. doi: 10.1210/endo-125-4-2092.
Numerous reports have appeared in the literature indicating phenotypic heterogeneity among cells of the osteoblastic lineage. This diversity may be due to either certain stages of differentiation or a subspecialization of already terminally differentiated osteoblasts. To obtain answers to this question, we report on studies undertaken to clone bone cell populations from 1 day postnatal rat calvaria which express well defined differences in phenotype. To achieve this goal, we have used the soft agarose cloning technique which previously has almost exclusively been applied to clone cells of neoplastic origin. The reason for being able to employ this method is based on the fact that bone cells can be induced by transforming growth factor-beta to reversibly acquire the transformed phenotype, an event expressed by anchorage-dependent bone cells to form progressively growing colonies in soft agarose. Individual colonies, harvested from agarose, were expanded to clonal bone cell populations. Characterizing 48 cell clones by detection of osteoblastic cell markers such as alkaline phosphatase activity, PTH- and prostaglandin-E2-induced adenylate cyclase activity, osteocalcin mRNA synthesis, as well as collagen synthesis, 7 subsets of osteoblastic cell types were identified. Each subset was found to express a distinct phenotype, indicated by the absence or presence of osteoblastic cell markers. Some clones, previously found not to exhibit any osteoblastic traits, developed PTH responsiveness when treated with insulin-like growth factor-I/transforming growth factor-beta, suggesting that these clones may originate from the osteoprogenitor cell pool. While most clonal cell populations were characterized as fully functional osteoblastic cells, some clones expressed merely 1, 2, or 3 osteoblastic markers, which suggests that they may represent stages of differentiation along the osteogenic pathway. In addition, other subclones displayed the capacity to synthesize osteocalcin and showed PTH and prostaglandin-E2 responsiveness, but were found to be devoid of alkaline phosphatase activity. Others expressed all osteoblastic cell markers except PTH responsiveness. The phenotypic constellation of the latter suggests that these cell clones may represent mature osteoblast-like cells, which, perhaps due to environmental circumstances present at the time of isolation, have become altered in accordance with the physiological requirements of the tissue.
文献中已有大量报道指出成骨细胞谱系细胞之间存在表型异质性。这种多样性可能是由于分化的某些阶段，或者是已经终末分化的成骨细胞的亚专业化。为了回答这个问题，我们报告了从出生后1天的大鼠颅骨中克隆骨细胞群体的研究，这些细胞在表型上表现出明确的差异。为了实现这一目标，我们使用了软琼脂糖克隆技术，该技术以前几乎专门用于克隆肿瘤起源的细胞。能够采用这种方法的原因基于这样一个事实，即骨细胞可以被转化生长因子-β诱导，可逆地获得转化表型，这是一种由贴壁依赖的骨细胞在软琼脂糖中形成逐渐生长的集落所表达的事件。从琼脂糖中收获的单个集落被扩增为克隆骨细胞群体。通过检测成骨细胞标记物，如碱性磷酸酶活性、甲状旁腺激素和前列腺素-E2诱导的腺苷酸环化酶活性、骨钙素mRNA合成以及胶原蛋白合成，对48个细胞克隆进行表征，确定了7个成骨细胞类型亚群。发现每个亚群都表达独特的表型，这通过成骨细胞标记物的存在或缺失来表明。一些以前发现不表现出任何成骨特征的克隆，在用胰岛素样生长因子-I/转化生长因子-β处理后产生了甲状旁腺激素反应性，这表明这些克隆可能起源于骨祖细胞池。虽然大多数克隆细胞群体被表征为功能完全的成骨细胞，但一些克隆仅表达1、2或3种成骨标记物，这表明它们可能代表成骨途径中的分化阶段。此外，其他亚克隆显示出合成骨钙素的能力，并表现出甲状旁腺激素和前列腺素-E2反应性，但被发现缺乏碱性磷酸酶活性。其他克隆表达了除甲状旁腺激素反应性之外的所有成骨细胞标记物。后者的表型组合表明，这些细胞克隆可能代表成熟的成骨样细胞，可能由于分离时存在的环境情况，已根据组织的生理需求发生了改变。
