Suppression of embroyoid body teratogenesis following in vivo transplantation of EB on a Honeycomb Sponge scaffold.
Mouse kidney at 12 weeks following transplantation of EB on KOU-CSH-10 Honeycomb Sponge scaffold (EB/+CSH) showed no sign of teratoma formation while EB transplanted without KOU-CSH-10 (ES/-CSH) generated teratomas in all mice. Histologically, transplanted ES/+CSH were indistinguishable from adjacent host renal tissue, suggesting spontaneous differentiation without specific induction.
Methods: Plate cultured mouse embryonic stem (ES) cells were trypsinized, filtered through nylon mesh, seeded into 96-well plates (1x104 cells/well), and cultured for 5 days to form embryoid bodies (EB). When EB were mixed with Honeycomb Sponge (KOU-CSH-10), EB rapidly and uniformly integrated into the KOU-CSH-10 matrix.
EB/+CSH complex was then transplanted under renal fascia of 6-week-old mice.
Atelocollagen Honeycomb sponge
Atelocollagen Honeycomb Disc 96
< Research brief >
We hypothesized that a combined use of ES cells and tooth mesenchymal cells is promising or successful development into tooth germ and/or teeth. Based on this hypothesis, we solated mesenchymal cells from fetal mice, and established a stable cell line, called MDU1 ine. We next in vitro cultured ES cells on MDU1 cells, which were used as feeder cells in the ulture. Then we found that the ES cells developed into cells that produced keratin and the DU1 cells into fibroblast-like cells that produced collagen. We therefore considered the cells hus co-cultured capable of developing into tooth germ and/or teeth. We also found that a mixture of cells co-cultured (ES and MDU1 cells) formed embryoid body (EB)-like spheres, called chimera Ebs. We next transplanted chimera Ebs with 3D scaffold made of type I collagen (called collagen sponge, CS) and transplanted the Ebs into sub-renal capsule in mice. Our close observations with histology and immuno-histochemistry identified extensive calcification, and significant development of odontoblasts and ameloblasts in the grafts. This identification indicates high potential of these Ebs as a good source for tooth germ and/or teeth, and therefore suggests that the absence of the entire structures is due to a small number of cells transplanted. Based on this suggestion, we plan to transplant a larger number of cells co-cultured to identify whether the cells develop into entire structures of tooth germ and/or teeth.
We have observed during the course of the above experiments that ES cells did not form teratoma when transplanted with CS for 7 weeks, whereas ES cells readily formed teratoma when transplanted alone. This observation is extremely important because teratoma formation is one of the most critical problems in practicing stem cell therapy and no perspective for solving this problem has been reported so far. To obtain a better understanding of this phenomenon, we examined gene expression of ES cells, and identified
expression of both undifferentiation genes and a tumor marker gene were significantly suppressed when the ES cells were co-cultured with CS. This suggests that CS can directly suppress teratoma formation, and also implies a good method to suppress teratoma formation from cells that experience specific induction from ES cells.
To be used for research only. DO NOT use for human gene therapy or clinical diagnosis.