Abstract
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.
Highlights
Embryonic stem (ES) cells are cells derived from early embryos that have not started to differentiate
We examine the development of fli:green fluorescence protein (GFP)+ and kdrl:GFP+ cells in the embryoid bodies (EBs) derived from zebrafish blastocyst cell cultures
Effect of medium composition on quantification of fli:GFP+ cells The blastocyst cells isolated from fli:GFP embryos showed expression of the fli:GFP marker in cells in culture medium with or without endothelial growth supplements
Summary
Embryonic stem (ES) cells are cells derived from early embryos that have not started to differentiate. By specific in vitro manipulation, these ES cells can maintain their growth and pluripotency (the ability to differentiate into multiple cell types) almost indefinitely [1]. Pluripotent ES cells can differentiate into specific cell types according to the culture conditions. Examples of differentiated cell types derived in vitro from ES cells include human cardiomyocytes [6], human neural progenitor cells [7], mouse hematopoietic progenitor cells [8], and alveolar epithelial cells [9]. One of the important cell types derived from embryonic stem cell culture is endothelial progenitor cells which form the epithelial lining of the cardiovascular system [10]
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