Large-scale production of murine embryonic stem cell-derived osteoblasts and chondrocytes on microcarriers in serum-free media
Large-scale production of murine embryonic stem cell-derived osteoblasts and chondrocytes on microcarriers in serum-free media
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DOI：
10.1016/j.biomaterials.2011.04.015
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发表时间：
2011-09-01
期刊：
影响因子：
14
通讯作者：
Kallos, Michael S.
中科院分区：
文献类型：
--
作者：
Alfred, Roz;Taiani, Jaymi T.;Kallos, Michael S.
从干细胞产生组织工程化结构用于治疗肌肉骨骼疾病可能会对再生医学产生巨大影响，但干细胞培养和分化存在困难，包括使用血清。在这里，我们提出了在搅拌悬浮生物反应器中成功地在CultiSpher S大孔微载体上生产小鼠胚胎干细胞来源的成骨细胞和软骨细胞的无血清方案。考察了不同接种物形式和搅拌速度。将产生的成骨细胞异位植入SCID小鼠体内，并原位植入小鼠钻孔骨折模型。Osterix、骨钙素和I型胶原在成骨培养中上调，聚集素和II型胶原在软骨培养中上调。茜素红S染色、von Kossa染色和艾氏蓝染色的组织学分析证实，培养的微载体和切除的组织中分别存在成骨细胞和软骨细胞。最后，将衍生细胞移植到小鼠骨折模型中，发现细胞整合，没有任何肿瘤形成。总体而言，微载体可以在体内植入的无血清生物过程中为胚胎干细胞的扩增和分化提供支持支架。这些发现为利用hESCs和iPS细胞开发肌肉骨骼损伤和疾病的临床治疗奠定了基础。(C)2011爱思唯尔有限公司。保留所有权利。
The generation of tissue-engineered constructs from stem cells for the treatment of musculoskeletal diseases may have immense impact in regenerative medicine, but there are difficulties associated with stem cell culture and differentiation, including the use of serum. Here we present serum-free protocols for the successful production of murine embryonic stem cell (mESC) derived osteoblasts and chondrocytes on CultiSpher S macroporous microcarriers in stirred suspension bioreactors. Various inoculum forms and agitation rates were investigated. Produced osteogenic cells were implanted ectopically into SCID mice and orthotopically into a murine burr-hole fracture model. Osterix, osteocalcin and collagen type I were upregulated in osteogenic cultures, while aggrecan and collagen type II were upregulated in chondrogenic cultures. Histological analysis using alizarin red S, von Kossa and alcian blue staining confirmed the presence of osteoblasts and chondrocytes, respectively in cultured microcarriers and excised tissue. Finally, implantation of derived cells into a mouse fracture model revealed cellular integration without any tumor formation. Overall, microcarriers may provide a supportive scaffold for ESC expansion and differentiation in a serum-free bioprocess for in vivo implantation. These findings lay the groundwork for the development of clinical therapies for musculoskeletal injuries and diseases using hESCs and iPS cells. (C) 2011 Elsevier Ltd. All rights reserved.