Institute of Biology Leiden, Leiden University, The Netherlands; Animal Biotechnology Division, Institute of Biotechnology and Genetic Engineering, The University of Agriculture Peshawar, Pakistan.
Institute of Biology Leiden, Leiden University, The Netherlands.
Exp Cell Res. 2020 Jul 15;392(2):112032. doi: 10.1016/j.yexcr.2020.112032. Epub 2020 Apr 28.
There is increasing interest in the possibility of culturing organ-like tissues (organoids) in vitro for biomedical applications. The ability to culture organoids would be greatly enhanced by having a functional circulation in vitro. The endothelial cell is the most important cell type in this context. Endothelial cells can be derived from pluripotent embryonic blastocyst cells in aggregates called embryoid bodies. Here, we examine the yield of endothelial-like cells in embryoid bodies (EBs) developed from transgenic zebrafish fli:GFP and kdrl:GFP blastocyst embryos. The isolated blastocyst cells developed into EBs within the first 24 h of culture and contained fli:GFP (putative endothelial, hematopoietic and other cell types); or kdrl:GFP (endothelial) cells. The addition of endothelial growth supplements to the media and culture on collagen type-I substratum increased the percentages of fli:GFP and kdrl:GFP cells in culture. We found that EBs developed in hanging-drop cultures possessed a higher percentage of fli:GFP (45.0 ± 3.1%) and kdrl:GFP cells (8.7 ± 0.7%) than those developed on conventional substrata (34.5 ± 1.4% or 5.2 ± 0.4%, respectively). The transcriptome analysis showed a higher expression of VEGF and TGFβ genes in EB cultures compared to the adherent cultures. When transferred to conventional culture, the percentage of fli:GFP or kdrl:GFP cells declined significantly over subsequent days in the EBs. The fli:GFP cells formed a monolayer around the embryoid bodies, while the kdrl:GFP cells formed vascular network-like structures in the embryoid bodies. Differences were observed in the spreading of fli:GFP cells, and network formation of kdrl:GFP cells on different substrates. The fli:GFP cells could be maintained in primary culture and sub-cultures. By contrast, kdrl:GFP cells were almost completely absent at 8d of primary culture. Our culture model allows real-time observation of fli:GFP and kdrl:GFP cells in culture. The results obtained from this study will be important for the development of vascular and endothelial cell culture using embryonic cells.
人们对在体外培养类器官（organoids）用于生物医学应用的可能性越来越感兴趣。如果能够在体外建立功能性循环，那么培养类器官的能力将会大大提高。在这种情况下，内皮细胞是最重要的细胞类型。内皮细胞可以从多能胚胎囊胚细胞聚集物（称为胚状体）中获得。在这里，我们检查了转基因斑马鱼 fli:GFP 和 kdrl:GFP 囊胚胚胎衍生的胚状体 (EBs) 中内皮样细胞的产量。在培养的头 24 小时内，分离的囊胚细胞发育成 EBs，其中包含 fli:GFP（假定的内皮细胞、造血细胞和其他细胞类型）；或 kdrl:GFP（内皮细胞）。在培养基中添加内皮生长补充剂并在胶原 I 基质上培养可增加培养物中 fli:GFP 和 kdrl:GFP 细胞的百分比。我们发现，在悬滴培养中发育的 EBs 具有更高百分比的 fli:GFP（45.0±3.1%）和 kdrl:GFP 细胞（8.7±0.7%），而在常规基质上发育的 EBs 则分别为 34.5±1.4%或 5.2±0.4%。转录组分析显示，EB 培养物中 VEGF 和 TGFβ 基因的表达水平高于贴壁培养物。当转移到常规培养时，EB 中 fli:GFP 或 kdrl:GFP 细胞的百分比在随后的几天中显著下降。fli:GFP 细胞在胚状体周围形成单层，而 kdrl:GFP 细胞在胚状体中形成血管网络样结构。在不同的基质上观察到 fli:GFP 细胞的铺展和 kdrl:GFP 细胞的网络形成存在差异。fli:GFP 细胞可以在原代培养和传代培养中维持。相比之下，在原代培养的第 8 天，kdrl:GFP 细胞几乎完全不存在。我们的培养模型允许实时观察培养物中的 fli:GFP 和 kdrl:GFP 细胞。本研究的结果对于使用胚胎细胞培养血管和内皮细胞将非常重要。
