Embryonic stem cells (ESCs) and neural progenitor cells form three-dimensional (3D) colonies or spheres in vitro, and 3D sphere is reported to help maintaining the stemness of stem cells, but the effect of 3D sphere formation on cell reprogramming remains unknown. Here we examined whether 3D sphere culture have any impact on the differentiated cells. We cultured bladder cancer cell RT4 and non-cancerous cell HEK293 on the low attachment dishes coated with soft agarose. When grown on this low attachment dish, cells spontaneously aggregated to form 3D spheres. Data showed that 3D sphere formation promoted the expression of reprogramming factors. Sphere formation of RT4 cells induced cancer stem cell characteristics including higher SP cell percentage, higher metastasis ability and higher tumorigenicity. HEK293 spheres showed upregulation of kidney progenitor cell markers and partially acquired characteristics of ESCs including upregulation of alkaline phosphatase activity, ES cell markers, three germ layer markers and tumorigenicity. The findings suggested that forced growth into 3D spheres by the low attachment surface could induce cells to acquire stemness properties.

Keywords:
Stemness; Reprogramming; 3D sphere culture; Low attachment surface