Dissociation of embryonic kidneys followed by reaggregation allows the formation of renal tissues.
Here we describe a novel method in which embryonic kidneys are dissociated into single-cell suspensions and then reaggregated to form organotypic renal structures. Kidney cell reaggregates were transiently cultured with small-molecule Rho kinase inhibitors, which caused ureteric bud structures to form and induced formation of nephrons. These structures displayed normal morphology, expressed appropriate differentiation markers, and were connected at their distal ends to the ureteric buds, thus forming artificial tissues very similar to those found in normal embryonic kidneys. Using this culture method, it was straightforward to make fine-grained chimeras by mixing different cell types or by mixing cells transfected with different constructs before reaggregation. Chimeric renal cultures were formed using mixtures of unmarked normal host embryonic kidney cells and CellTracker-marked WT1 siRNA-carrying cells to test the hypothesis that WT1 is important to a cell's ability to contribute to nephron formation. We found a significant reduction in the ability of WT1 knockdown cells to contribute to nephron formation. This dissociation and reaggregation procedure can also be applied to embryonic lungs and to form coarse-grained hybrid tissues from mixtures of lung and kidney cells. Overall, our protocol allows very simple mixing of cells from different sources or cells subjected to different pretreatments to make fine-grained, highly dispersed chimera tissues.
Unbekandt
Mathieu
M
Centre for Integrative Physiology, University of Edinburgh, Edinburgh, UK.
Davies
Jamie A
JA
D000818:Animals; D002449:Cell Aggregation / Q000235:genetics; D004622:Embryo, Mammalian / Q000166:cytology / Q000378:metabolism; D004847:Epithelial Cells / Q000378:metabolism; D007150:Immunohistochemistry; D007668:Kidney / Q000166:cytology* / Q000196:embryology*; D007685:Kidney Tubules, Collecting / Q000196:embryology / Q000378:metabolism*; D051379:Mice; D008954:Models, Biological; D009399:Nephrons / Q000196:embryology / Q000378:metabolism*; D009924:Organ Culture Techniques; D034741:RNA, Small Interfering / Q000378:metabolism; D014162:Transfection; D051153:Wnt Proteins / Q000378:metabolism
D034741:RNA, Small Interfering; D051153:Wnt Proteins