Bone matrix formation in osteogenic cultures derived from human embryonic stem cells in vitro
Bone matrix formation in osteogenic cultures derived from human embryonic stem cells in vitro
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DOI：
10.1089/scd.2006.0010
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发表时间：
2007-02-01
影响因子：
4
通讯作者：
Wendel, Mikael
中科院分区：
文献类型：
--
作者：
Karner, Elerin;Unger, Christian;Wendel, Mikael
骨基质的产生和矿化涉及复杂的机制，包括有机细胞外基质的初始形成，无机羟基磷灰石晶体随后沉积到该有机细胞外基质中。人类胚胎干细胞（hES）提供了研究早期发育过程的潜力，并提供了无限的细胞来源。在这项研究中，使用了四种不同的hES细胞系，并采取了两种不同的方法将hES细胞分化为成骨谱系。未分化的细胞培养悬浮液中，促进胚状体（EB）的形成，或在单层，这两种方法都在成骨补充剂的存在下。我们的成骨分化研究的新方法是使用市售的人包皮成纤维细胞来支持hES细胞集落的未分化生长，以及它们在含血清促分化培养基中的增殖。成骨表型的表征显示，所有的hES细胞系分化向间充质谱系，因为T-短尾畸形，Flt-1，和骨形态发生蛋白-4可以检测到。主要成骨细胞标志基因Runx 2、osterix、骨涎蛋白、骨钙素表达上调。茜素红S染色显示骨样结节形成，免疫组化显示骨唾液蛋白和骨钙素定位于这些病灶。在单层条件下分化的细胞表现出更大的成骨潜力相比，EB衍生的细胞。我们的结论是，在体外hES细胞可以产生一个矿化的基质拥有所有主要的骨标志物，多能hES细胞的分化成骨细胞系不需要通过EB形成的启动，和谱系的潜力是不依赖于分化诱导的模式，但对细胞系本身。
Bone matrix production and mineralization involves sophisticated mechanisms, including the initial formation of an organic extracellular matrix into which inorganic hydroxyapatite crystals are later deposited. Human embryonic stem (hES) cells offer a potential to study early developmental processes and provide an unlimited source of cells. In this study, four different hES cell lines were used, and two different approaches to differentiate hES cells into the osteogenic lineage were taken. Undifferentiated cells were cultured either in suspension, facilitating the formation of embryoid bodies (EBs), or in monolayer, and both methods were in the presence of osteogenic supplements. Novel to our osteogenic differentiation study was the use of commercially available human foreskin fibroblasts to support the undifferentiated growth of the hES cell colonies, and their propagation in serum replacement-containing medium. Characterization of the osteogenic phenotype revealed that all hES cell lines differentiated toward the mesenchymal lineage, because T-Brachyury, Flt-1, and bone morphogenetic protein-4 could be detected. Main osteoblastic marker genes Runx2, osterix, bone sialoprotein, and osteocalcin were up-regulated. Alizarin Red S staining demonstrated the formation of bone-like nodules, and bone sialoprotein and osteocalcin were localized to these foci by immunohistochemistry. Cells differentiated in monolayer conditions exhibited greater osteogenic potential compared to those from EB-derived cells. We conclude that in vitro hES cells can produce a mineralized matrix possessing all the major bone markers, the differentiation of pluripotent hES cells to an osteogenic lineage does not require initiation via EB formation, and that lineage potential is not dependent on the mode of differentiation induction but on a cell line itself.