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Somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs), which have promising potential applications in regenerative medicine. However, the challenges of successful applications of human iPSCs for medical purposes are the low generation efficiency, heterogeneous colonies, and exposure to the animal-derived product Matrigel. We aimed to investigate whether human urinal cells could be efficiently reprogrammed into iPSCs in three-dimensional Puramatrix (3D-PM) compared to two-dimensional Matrigel (2D-MG) and to understand how this 3D hydrogel environment affects the reprogramming process. Human urinal cells were successfully reprogrammed into iPSCs in the defined synthetic animal-free 3D-PM. Interestingly, although the colony efficiency in 3D-PM was similar to that in 2D-MG (∼0.05%), the reprogrammed colonies in 3D-PM contained an iPSC population with significantly higher homogeneity, as evidenced by the pluripotent-like morphology and expression of markers. This was further confirmed by transcriptome profile analysis in bulk cells and at the single cell level. Moreover, the homogeneity of the iPSC population in 3D-PM colonies was correlated with the downregulation of integrin β1 (ITGB1) and phosphorylated focal adhesion kinase (FAK). Collectively, 3D-PM provides an alternative approach for obtaining iPSCs with enhanced homogeneity. This work also unveiled the regulation of human somatic cell reprogramming via the extracellular microenvironment.

The use of differentiating human induced pluripotent stem cells (hiPSCs) in mini-tissue organoids provides an invaluable resource for regenerative medicine applications, particularly in the field of disease modeling. However, most studies using a kidney organoid model, focused solely on the transcriptomics and did not explore mechanisms of regulating kidney organoids related to metabolic effects and maturational phenotype. Here, we applied metabolomics coupled with transcriptomics to investigate the metabolic dynamics and function during kidney organoid differentiation. Not only did we validate the dominant metabolic alteration from glycolysis to oxidative phosphorylation in the iPSC differentiation process but we also showed that glycine, serine, and threonine metabolism had a regulatory role during kidney organoid formation and lineage maturation. Notably, serine had a role in regulating S-adenosylmethionine (SAM) to facilitate kidney organoid formation by altering DNA methylation. Our data revealed that analysis of metabolic characterization broadens our ability to understand phenotype regulation. The utilization of this comparative omics approach, in studying kidney organoid formation, can aid in deciphering unique knowledge about the biological and physiological processes involved in organoid-based disease modeling or drug screening.