In vitro differentiation and in vivo mineralization of osteogenic cells derived from human embryonic stem cells
In vitro differentiation and in vivo mineralization of osteogenic cells derived from human embryonic stem cells
复制标题
DOI：
10.1089/ten.2004.10.1518
复制
发表时间：
2004-09-01
影响因子：
--
通讯作者：
Buttery, LDK
中科院分区：
文献类型：
--
作者：
Bielby, RC;Boccaccini, AR;Buttery, LDK
人类胚泡来源胚胎干细胞系的首次报道对发育生物学和再生医学的研究具有重要意义。寻找有效和可重复的方法从ES细胞来源中获得治疗有用的细胞是许多再生医学策略的关键特征。我们之前已经证明，在培养液中添加抗坏血酸、β-甘油磷酸和地塞米松可以诱导小鼠ES细胞成骨分化。这项研究探讨了用小鼠ES细胞开发的促进成骨分化的方法是否能成功地应用于人ES细胞。H1系在小鼠饲养层上体外繁殖，通过标记Oct-4和SSEA-4的表达证明其具有多能性。随后，通过类胚体(EB)的形成启动分化，在悬浮培养5天后，从EBS中获得的细胞在含有成骨补充剂的培养液中复制。我们发现，先前被认为对小鼠ES细胞最佳的治疗方案，特别是在特定时间点添加地塞米松，也能诱导人类ES细胞产生最大的成骨反应。我们在体外鉴定了骨钙素免疫染色阳性的矿化细胞，并发现必需的骨转录因子Runx2的表达增加。当移植到SCID小鼠体内的聚-D，L-丙交酯(PDLLA)支架上，细胞具有在体内形成矿化组织的能力。植入35天后，通过von Kossa染色和人类形式骨钙素的免疫表达，可以在支架内识别矿化组织区域。我们没有看到任何畸胎瘤形成的证据。因此，这些数据表明，成骨细胞来源于多能的人类ES细胞，在体外和体内都具有形成矿化组织的能力。我们还表明，为小鼠ES细胞分化建立的培养方法完全可以移植到人类ES细胞上。这项技术的进一步发展将导致产生足够产量的成骨细胞用于骨骼组织修复的能力。
The first report of the derivation of embryonic stem (ES) cell lines from human blastocysts had major implications for research into developmental biology and regenerative medicine. Finding efficient and reproducible methods to derive therapeutically useful cells from an ES cell source is a key feature of many regenerative medicine strategies. We have previously demonstrated that it is possible to induce osteogenic differentiation of murine ES cells by supplementing the culture medium with ascorbic acid, beta-glycerophosphate, and dexamethasone. This study investigated whether methods for driving osteogenic differentiation developed with murine ES cells could be applied successfully to human ES cells. The H1 line was propagated in vitro on murine feeder layers and shown to be pluripotent by expression of the markers Oct-4 and SSEA-4. Subsequently, differentiation was initiated via embryoid body (EB) formation and, after 5 days in suspension culture, cells harvested from EBs were replated in a medium containing osteogenic supplements. We found that the treatment regimen previously identified as optimal for murine ES cells, and in particular the addition of dexamethasone at specific time points, also induced the greatest osteogenic response from human ES cells. We identified mineralizing cells in vitro that immunostained positively for osteocalcin and found an increase in expression of an essential bone transcription factor, Runx2. When implanted into SCID mice on a poly-D, L-lactide (PDLLA) scaffold, the cells had the capacity to give rise to mineralized tissue in vivo. After 35 days of implantation, regions of mineralized tissue could be identified within the scaffold by von Kossa staining and immunoexpression of the human form of osteocalcin. We did not see any evidence of teratoma formation. These data therefore demonstrate the derivation of osteoblasts from pluripotent human ES cells with the capacity to form mineralized tissue both in vitro and in vivo. We have also shown that a culture methodology established for differentiation of murine ES cells was entirely transferable to human ES cells. Further development of this technology will result in the capacity to generate sufficient yields of osteogenic cells for use in skeletal tissue repair.