Biological Systems Engineering Laboratory, Department of Chemical Engineering and Chemical Technology, Imperial College London, London, United Kingdom.
Stem Cells Dev. 2012 Nov 20;21(17):3152-61. doi: 10.1089/scd.2012.0030. Epub 2012 Aug 16.
Embryonic stem cell (ESC) differentiation via embryoid body (EB) formation is an established method that generates the 3 germ layers. However, EB differentiation poses several problems including formation of heterogeneous cell populations. Previously, we have enhanced mesoderm derivation from murine ESCs (mESCs) using conditioned medium (CM) from HepG2 cells. We used this technique to direct hematopoiesis by generating "embryoid-like" colonies (ELCs) from mESCs without standard formation of EBs. Two predifferentiation conditions were tested: (1) mESCs cultured 3 days in standard predifferentiation medium (control) and (2) mESCs cultured 3 days in HepG2 CM (CM-mESCs). Both groups were then exposed to primary differentiation for 8 days (ELC-formation period) and 14 days of hematopoietic differentiation. Enhanced mesoderm formation was observed in the CM-mESC group with an almost 5-fold increase in ELC formation (P ≤ 0.05) and higher expression of mesoderm genes-Brachyury-T, Goosecoid, and Flk-1-compared with those of control mESCs. Hematopoietic colony formation by CM-mESCs was also enhanced by 2-fold at days 7 and 14 with earlier colony commitment compared with those of control mESCs (P ≤ 0.05). This early clonogenic capacity was confirmed morphologically by the presence of nucleated erythrocytes and macrophages as early as day 7 in CM-mESC culture using standard 14-day colony-forming assay. Early expression of hematopoietic primitive (ζ-globin) and definitive (β-globin) erythroid genes and proteins was also observed by day 7 in CM-mESC cultures. These data indicate that hematopoietic cells more quickly differentiate from CM-mESCs, compared with those using standard EB approaches, and provide an efficient bioprocess platform for erythroid-specific differentiation of ESCs.
胚胎干细胞 (ESC) 通过胚体 (EB) 形成的分化是一种已建立的方法，可生成 3 个胚层。然而，EB 分化存在几个问题，包括形成异质细胞群体。以前，我们使用 HepG2 细胞的条件培养基 (CM) 增强了来自鼠胚胎干细胞 (mESC) 的中胚层的衍生。我们使用这项技术通过从不形成标准 EB 的 mESC 中生成“胚样”集落 (ELC) 来指导造血。测试了两种预分化条件：(1) mESC 在标准预分化培养基中培养 3 天（对照）和 (2) mESC 在 HepG2 CM 中培养 3 天（CM-mESC）。然后，两组均暴露于初级分化 8 天（ELC 形成期）和 14 天的造血分化。在 CM-mESC 组中观察到增强的中胚层形成，ELC 形成增加近 5 倍（P ≤ 0.05），并且中胚层基因-Brachyury-T、Goosecoid 和 Flk-1 的表达更高与对照 mESC 相比。CM-mESC 的造血集落形成也增强了 2 倍，在第 7 天和第 14 天的集落承诺早于对照 mESC（P ≤ 0.05）。通过使用标准的 14 天集落形成测定法，在 CM-mESC 培养物中，早在第 7 天就通过存在有核红细胞和巨噬细胞来确认这种早期克隆形成能力的形态。在 CM-mESC 培养物中也观察到早期表达造血原始（ζ-球蛋白）和确定（β-球蛋白）红细胞基因和蛋白质。这些数据表明，与使用标准 EB 方法相比，造血细胞更快地从 CM-mESC 分化，并为 ESC 的红细胞特异性分化提供了有效的生物过程平台。
