Abstract
In order to harness the potential of pluripotent stem cells, we need to understand how to differentiate them to our target cell types. Here, we developed a protocol to differentiate mouse embryonic stem cells (ESCs) to renal progenitors in a step-wise manner. Microarrays were used to track the transcriptional changes at each stage of differentiation and we observed that genes associated with metanephros, ureteric bud, and blood vessel development were significantly upregulated as the cells differentiated towards renal progenitors. Priming the ESCs and optimizing seeding cell density and growth factor concentrations helped improve differentiation efficiency. Organoids were used to determine the developmental potential of the renal progenitor cells. Aggregated renal progenitors gave rise to organoids consisting of LTL+/E-cadherin+ proximal tubules, cytokeratin+ ureteric bud-derived tubules, and extracellular matrix proteins secreted by the cells themselves. Over-expression of key kidney developmental genes, Pax2, Six1, Eya1, and Hox11 paralogs, during differentiation did not improve differentiation efficiency. Altogether, we developed a protocol to differentiate mouse ESCs in a manner that recapitulates embryonic kidney development and showed that precise gene regulation is essential for proper differentiation to occur.
Highlights
Pluripotent stem cells (PSCs), such as embryonic stem cells (ESCs) and induced PSCs, have the ability to self-renew and differentiate into all cells of all three germ layers, including renal progenitors
When cultured under conditions that mimic embryonic kidney development, PSCs can be differentiated to renal progenitors and coaxed to form kidney organoids, and these cells can be used in various applications, including cell-based therapy and nephrotoxicity screening
Using quantitative polymerase chain reaction (PCR), we showed E13.5 embryonic kidney would reveal genes differentially that our differentiation protocol yields a mix of renal progenitors, expressed between the samples and indicate what genes are including metanephric mesenchyme (Osr[1], Sall1), nephric duct lacking in each sample with respect to the other
Summary
Pluripotent stem cells (PSCs), such as embryonic stem cells (ESCs) and induced PSCs (iPSCs), have the ability to self-renew and differentiate into all cells of all three germ layers, including renal progenitors. And quantitative PCR was used to show that pluripotency- focusing on 190 metanephric mesenchyme-associated genes and associated markers Esrrb, Lefty[2], and Lin28a were significantly 298 ureteric bud-associated genes, the gene expression profiles of downregulated in progenitors compared to undifferentiated ESCs renal progenitors were more similar to E12.5/13.5 mouse (Supplementary Fig. 3) This indicated that the differentiated renal embryonic kidneys, undifferentiated ESC, mesoderm cells, progenitors are not pluripotent. This indicates that the organoids whether the over-expression of Six[1], Pax[2], Eya[1], Hoxa[11], Hoxc[11], are more similar to mouse embryonic kidneys than adult kidneys, and Hoxd[11] in mouse ESCs would improve renal differentiation but has commonality with the adult kidney for some of the efficiency and possibly maturation. Our data suggest that over-expression of Six[1], Pax[2], Eya[1], Hoxa[11], Hoxc[11], and Hoxd[11] inhibits renal differentiation
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