Dissociation of Embryonic Kidney Followed by Re-aggregation as a Method for Chimeric Analysis

Abstract

Dissociation of embryonic kidney followed by re-aggregation as a method for chimeric analysis. General rightsCopyright for the publications made accessible via the Edinburgh Research Explorer is retained by the author(s) and / or other copyright owners and it is a condition of accessing these publications that users recognise and abide by the legal requirements associated with these rights. Take down policyThe University of Edinburgh has made every reasonable effort to ensure that Edinburgh Research Explorer content complies with UK legislation. If you believe that the public display of this file breaches copyright please contact openaccess@ed.ac.uk providing details, and we will remove access to the work immediately and investigate your claim. Summary:This chapter presents three methods for re---constructing mouse foetal kidney tissue from simple suspensions of cells. These techniques are very useful for a number of purposes; (i) they allow the production of fine---grained chimaeras in which cell autonomy of mutations can be tested, (ii) they provide an environment allow the renal differentiation potential of stem cells to be assessed, and (iii) they are an excellent system in which to study the mechanisms of self---organization. Each of the methods described here begins with disaggregation of embryonic mouse kidneys, followed by re---aggregation and culture; the main differences are in the culture methods, each of which has advantages for particular purposes.

Self-organisation after embryonic kidney dissociation is driven via selective adhesion of ureteric epithelial cells.

“…Each aggregate, comprising 4×10 5 centrifuged single cells, was cultured on a Transwell membrane insert (Sigma) in a 6-well plate containing 700 µl of DMEM (Life Technologies) plus 10% foetal bovine serum (Scientifix), and 1% penicillin-streptomycin (Life Technologies). Unlike previously reported protocols (Davies et al, 2012), aggregates were not cultured in the presence of ROCKi (designed to reduce cell death) or on a monolayer of Wnt-expressing NIH3T3 (Lusis et al, 2010). We did not find that either of these features was required when commencing with 13.0 dpc embryonic kidneys.…”

“…All antibody staining was performed as previously described (Davies et al, 2012). In brief, the 4% paraformaldehyde-fixed and PBS-washed aggregates were excised from the Transwell membrane filter individually with a scalpel and forceps.…”

“…1B). After 4-6 days of culture, the resulting aggregates reform collecting duct epithelium surrounded by nephron progenitors that give rise to nascent nephrons (Unbekandt and Davies, 2010;Lusis et al, 2010;Davies et al, 2012;Hendry et al, 2013). This approach of dissociation and reaggregation of embryonic tissue serves as an appropriate experimental system within which to investigate how this occurs.…”

Rapid and Efficient Differentiation of Human Pluripotent Stem Cells into Intermediate Mesoderm That Forms Tubules Expressing Kidney Proximal Tubular Markers

“…43 Cells expressing IM markers were dissociated on day 3 (PAX2 + LHX1 + ) or 9 (LTL + KSP + ) of differentiation and were recombined with dissociated cells from wild-type E12.5 mouse embryonic kidneys. Human cells from day 3 were incorporated into mouse metanephric tissues, distributing in the interstitium; however, no tubular integration was observed.…”

“…43,66 Briefly, embryonic kidneys at stage E12.5 (day of plug=E0.5) were isolated from timed pregnant females (Charles River).…”