Differentiation of pluripotent stem cells to form renal organoids
A method is provided for producing renal organoids comprising nephrons, ureteric bud and vasculature and/or progenitors of these. In one embodiment, the methods includes contacting intermediate mesoderm cells with: fibroblast growth factor 9 and/or fibroblast growth factor 20 and/or fibroblast growth factor 2 and optionally, one or more selected from the group consisting of: bone morphogenic protein 7; heparin; a Wnt agonist; retinoic acid; and an RA antagonist under conditions that promote formation of vascularized renal organoids. Another embodiment includes producing mesoderm cells by sequentially contacting pluripotent stem cells with a Wnt agonist and fibroblast growth factor 9 and/or fibroblast growth factor 20 and/or fibroblast growth factor 2, followed by a relatively short re-exposure to the Wnt agonist. The renal organoids may have end uses such as for kidney repair and regeneration, bioprinting of kidneys or functional components thereof, renal cell arrays and screening compounds for nephrotoxicity.
1. A method of producing one or more renal organoids comprising:
(a) differentiating intermediate mesoderm (IM) cells into nephron progenitor cells and ureteric epithelial progenitor cells by contacting the IM cells with a Wnt agonist, fibroblast growth factor 9 (FGF9), and one or more compounds selected from the group consisting of bone morphogenic protein 7 (BMP7), heparin, retinoic acid (RA), an RA analog, an RA agonist, and an RA antagonist, wherein the RA antagonist increases the relative production of nephron progenitor cells from the IM cells; and
(b) culturing the nephron progenitor cells and ureteric epithelial progenitor cells under conditions that induce aggregation of the nephron progenitor cells and ureteric epithelial progenitor cells into one or more renal organoids that are at least partly vascularized and/or comprise vascular progenitors, wherein the vascularization is facilitated by conditions that promote or direct development of vascular endothelium or vascular progenitors from mesenchymal cells or tissues.
2. The method of claim 1 , wherein the RA analog or the RA agonist increases the relative production of ureteric epithelial progenitor cells from the IM cells.
3. The method of claim 1 , wherein the Wnt agonist increases the relative production of nephron progenitor cells from the IM cells.
4. The method of claim 1 , wherein the nephron progenitor cells and ureteric epithelial progenitor cells are produced synchronously or simultaneously from the IM cells.
5. The method of claim 1 , further comprising contacting posterior primitive streak cells with one or more agents that facilitate differentiation of the posterior primitive streak cells into said IM cells.
6. The method of claim 5 , further comprising contacting human pluripotent stem cells (hPSCs) with one or more agents that facilitate differentiation of the hPSCs into said posterior primitive streak cells.
7. The method of claim 1 , wherein the Wnt agonist is CHIR99021.
8. The method of claim 1 , wherein the IM cells are (i) first contacted with the Wnt agonist and (ii) then contacted with the FGF9.
9. The method of claim 8 , wherein the Wnt agonist is CHIR99021.
10. The method of claim 8 , further comprising (iii) contacting the cells resulting from step (ii) with a Wnt agonist.
11. The method of claim 10 , wherein the Wnt agonist in step (i) is CHIR99021.
12. The method of claim 10 , wherein the Wnt agonist in step (iii) is CHIR99021.
13. The method of claim 10 , wherein the Wnt agonist in step (i) is CHIR99021 and the Wnt agonist in step (iii) is CHIR99021.
14. The method of claim 13 , wherein the concentration of the Wnt agonist in step (i) is about 1-20 μM, the concentration of FGF9 is about 100-300 ng/ml, and the concentration of the Wnt agonist in step (iii) is about 1-15 μM.
15. The method of claim 14 , wherein the concentration of the Wnt agonist in step (i) is 5-15 μM.
16. The method of claim 1 , wherein the one or more compounds is heparin.
17. The method of claim 16 , wherein the IM cells are (i) first contacted with the Wnt agonist and (ii) then contacted with the FGF9 and the heparin.
18. The method of claim 17 , wherein the Wnt agonist is CHIR99021.
19. The method of claim 17 , further comprising (iii) contacting the cells resulting from step (ii) with a Wnt agonist.
20. The method of claim 19 , wherein the Wnt agonist in step (i) is CHIR99021.
21. The method of claim 19 , wherein the Wnt agonist in step (iii) is CHIR99021.
22. The method of claim 19 , wherein the Wnt agonist in step (i) is CHIR99021 and the Wnt agonist in step (iii) is CHIR99021.
23. The method of claim 22 , wherein the concentration of the Wnt agonist in step (i) is about 1-20 μM, the concentration of FGF9 is about 100-300 ng/ml, and the concentration of the Wnt agonist in step (iii) is about 1-15 μM.
24. The method of claim 23 , wherein the concentration of the Wnt agonist in step (i) is 5-15 μM.