Subfractionation of differentiating human embryonic stem cell populations allows the isolation of a mesodermal population enriched for intermediate mesoderm and putative renal progenitors.
Human embryonic stem (ES) cells are pluripotent and are believed to be able to generate all cell types in the body. As such, they have potential applications in regenerative therapy for kidney disease. However, before this can be achieved, a protocol to differentiate human ES cells to mesodermal renal progenitor lineages is required. Reduction of serum concentration and feeder layer density reduction cultures were used to differentiate human ES cells for 14 days. Differentiated ES cells were then fractionated by flow cytometry based on expression of the markers CD24, podocalyxin, and GCTM2 to isolate putative renal cells. These cells up-regulated the expression of the renal transcription factors PAX2, LHX1, and WT1 when compared with unfractionated human ES cells. Immunohistochemical assays confirmed that a subset of cells within this fraction co-expressed nuclear WT1 and PAX2 proteins. Transcriptome profiling also showed that the most differentially up-regulated genes in this fraction preferentially associated with kidney development in comparison with any other lineage. When compared with a transcriptome profile database of urogenital development (GUDMAP), the top 200 differentially up-regulated genes in this fraction strongly clustered into a group of genes associated with the metanephric mesenchyme at E11.5 and the corticonephrogenic interstitium at E15.5 of murine kidney development. Hence, this approach confirms an ability to direct human ES cells toward a renal progenitor state.
Department of Anatomy and Developmental Biology, Monash University, Clayton, Victoria, Australia.
S Adelia
SA
Lin
Gabriel
G
Kolle
Sean M
SM
Grimmond
Qi
Q
Zhou
Elizabeth
E
Doust
Melissa H
MH
Little
Bruce
B
Aronow
Sharon D
SD
Ricardo
Martin F
MF
Pera
John F
JF
Bertram
Andrew L
AL
Laslett
D000818:Animals; D002454:Cell Differentiation; D019070:Cell Lineage; D002469:Cell Separation / Q000379:methods*; D002478:Cells, Cultured; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000502:physiology; D015870:Gene Expression; D006801:Humans; D007668:Kidney / Q000166:cytology / Q000196:embryology; D008297:Male; D008648:Mesoderm / Q000166:cytology*; D051379:Mice; D016513:Mice, SCID; D046228:Microarray Analysis; D033581:Stem Cell Transplantation; D013724:Teratoma / Q000378:metabolism / Q000473:pathology