Department of Biochemistry and Molecular Biology, University of Calgary, Calgary, Alberta, Canada.
PLoS One. 2010 Mar 12;5(3):e9663. doi: 10.1371/journal.pone.0009663.
Due to their self-renewal, embryonic stem cells (ESCs) are attractive cells for applications in regenerative medicine and tissue engineering. Although ESC differentiation has been used as a platform for generating bone in vitro and in vivo, the results have been unsatisfactory at best. It is possible that the traditional culture methods, which have been used, are not optimal and that other approaches must be explored.
METHODOLOGY/PRINCIPAL FINDINGS: ESCs were differentiated into osteoblast lineage using a micro-mass approach. In response to osteogenic differentiation medium, many cells underwent apoptosis, while others left the micro-mass, forming small aggregates in suspension. These aggregates were cultured in three different culture conditions (adhesion, static suspension, and stirred suspension), then examined for osteogenic potential in vitro and in vivo. In adhesion culture, ESCs primed to become osteoblasts recommitted to the adipocyte lineage in vitro. In a static suspension culture, resulting porous aggregates expressed osteoblasts markers and formed bone in vivo via intermembranous ossification. In a stirred suspension culture, resulting non-porous aggregates suppressed osteoblast differentiation in favor of expanding progenitor cells.
CONCLUSIONS/SIGNIFICANCE: We demonstrate that microenvironment modulates cell fate and subsequent tissue formation during ESC differentiation. For effective tissue engineering using ESCs, it is important to develop optimized cell culture/differentiation conditions based upon the influence of microenvironment.
由于具有自我更新能力，胚胎干细胞（ESC）是再生医学和组织工程中应用的理想细胞。尽管 ESC 分化已被用于体外和体内生成骨，但结果充其量只能说是差强人意。有可能是因为我们一直使用的传统培养方法不够理想，需要探索其他方法。
方法/主要发现：我们使用微团块方法将 ESC 分化为成骨细胞谱系。在对成骨分化培养基的反应中，许多细胞发生凋亡，而其他细胞则离开微团块，在悬浮液中形成小聚集体。这些聚集体在三种不同的培养条件（贴壁、静态悬浮和搅拌悬浮）中进行培养，然后在体外和体内检测其成骨潜能。在贴壁培养中，准备成为成骨细胞的 ESC 在体外重新定向为脂肪细胞谱系。在静态悬浮培养中，形成的多孔聚集体表达成骨细胞标志物，并通过膜内成骨在体内形成骨。在搅拌悬浮培养中，形成的非多孔聚集体抑制成骨细胞分化，有利于扩增祖细胞。
结论/意义：我们证明微环境在 ESC 分化过程中调节细胞命运和随后的组织形成。为了有效利用 ESC 进行组织工程，根据微环境的影响，开发优化的细胞培养/分化条件非常重要。
