The generation of organoids is one of the biggest scientific advances in regenerative medicine. Here, by lengthening the time that human pluripotent stem cells (hPSCs) were exposed to a three-dimensional microenvironment, and by applying defined renal inductive signals, we generated kidney organoids that transcriptomically matched second-trimester human fetal kidneys. We validated these results using ex vivo and in vitro assays that model renal development. Furthermore, we developed a transplantation method that utilizes the chick chorioallantoic membrane. This approach created a soft in vivo microenvironment that promoted the growth and differentiation of implanted kidney organoids, as well as providing a vascular component. The stiffness of the in ovo chorioallantoic membrane microenvironment was recapitulated in vitro by fabricating compliant hydrogels. These biomaterials promoted the efficient generation of renal vesicles and nephron structures, demonstrating that a soft environment accelerates the differentiation of hPSC-derived kidney organoids.

Fanconi Anemia (FA) is a recessive disorder characterized by genomic instability, congenital abnormalities, cancer predisposition and bone marrow failure. However, the pathogenesis of FA is not fully understood partly due to the limitations of current disease models. Here, we derive integration-free induced pluripotent stem cells (iPSCs) from an FA patient without genetic complementation and report in situ gene correction in FA-iPSCs as well as the generation of isogenic FANCA deficient human embryonic stem cell (ESC) lines. FA cellular phenotypes are recapitulated in iPSCs/ESCs and their adult stem/progenitor cell derivatives. By using isogenic pathogenic mutation-free controls as well as cellular and genomic tools, our model serves to facilitate the discovery of novel disease features. We validate our model as a drug-screening platform by identifying several compounds that improve hematopoietic differentiation of FA-iPSCs. These compounds are also able to rescue the hematopoietic phenotype of FA-patient bone marrow cells.

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MicroRNAs (miRNAs) play an important role in proper function and differentiation of mouse embryonic stem cells (ESCs). We performed a systematic comparison of miRNA expression in undifferentiated vs. differentiating ESCs. We report that 138 miRNAs are increased on the induction of differentiation. We compared the entire list of candidate mRNA targets of up-regulated miRNAs with that of mRNA down-regulated in ESCs on induction of differentiation. Among the candidate targets emerging from this analysis, we found three genes, Smarca5, Jarid1b, and Sirt1, previously demonstrated to be involved in sustaining the undifferentiated phenotype in ESCs. On this basis, we first demonstrated that Smarca5 is a direct target of miR-100, Jarid1b of miR-137, and we also confirmed previously published data demonstrating that Sirt1 is a direct target of miR-34a in a different context. The suppression of these three miRNAs by anti-miRs caused the block of ESC differentiation induced by LIF withdrawal. On the other hand, the overexpression of the three miRNAs resulted in an altered expression of differentiation markers. These results demonstrate that miR-34a, miR-100, and miR-137 are required for proper differentiation of mouse ESCs, and that they function in part by targeting Sirt1, Smarca5, and Jarid1b mRNAs.