Gene-Edited Human Kidney Organoids Reveal Mechanisms of Disease in Podocyte Development.

A critical event during kidney organogenesis is the differentiation of podocytes, specialized epithelial cells that filter blood plasma to form urine. Podocytes derived from human pluripotent stem cells (hPSC-podocytes) have recently been generated in nephron-like kidney organoids, but the developmental stage of these cells and their capacity to reveal disease mechanisms remains unclear. Here, we show that hPSC-podocytes phenocopy mammalian podocytes at the capillary loop stage (CLS), recapitulating key features of ultrastructure, gene expression, and mutant phenotype. hPSC-podocytes in vitro progressively establish junction-rich basal membranes (nephrin+ podocin+ ZO-1+ ) and microvillus-rich apical membranes (podocalyxin+ ), similar to CLS podocytes in vivo. Ultrastructural, biophysical, and transcriptomic analysis of podocalyxin-knockout hPSCs and derived podocytes, generated using CRISPR/Cas9, reveals defects in the assembly of microvilli and lateral spaces between developing podocytes, resulting in failed junctional migration. These defects are phenocopied in CLS glomeruli of podocalyxin-deficient mice, which cannot produce urine, thereby demonstrating that podocalyxin has a conserved and essential role in mammalian podocyte maturation. Defining the maturity of hPSC-podocytes and their capacity to reveal and recapitulate pathophysiological mechanisms establishes a powerful framework for studying human kidney disease and regeneration. Stem Cells 2017;35:2366-2378.

Optical tweezers system for live stem cell organization at the single-cell level.

Cell manipulation is one of the most impactful applications for optical tweezers, and derived from this promise, we demonstrate a new optical tweezers system for the study of cell adhesion and organization. This method utilizes photonic-crystal-enhanced optical tweezers to manipulate cells with low laser intensities. By doing so, it enables effective cell patterning and culturing within the conditions necessary for successful differentiation and colony formation of human pluripotent stem cells. To this end, the biocompatibility of plasma-treated parylene-C for cell culturing was studied, and a thorough characterization of cellular interactive forces was performed using this system. Furthermore, this study also demonstrates construction of patterned cell arrays at arbitrary positions with micrometer-scale precision.

Photonic Crystal Optical Tweezers with High Efficiency for Live Biological Samples and Viability Characterization.

We propose and demonstrate a new optical trapping method for single cells that utilizes modulated light fields to trap a wide array of cell types, including mammalian, yeast, and Escherichia coli cells, on the surface of a two-dimensional photonic crystal. This method is capable of reducing the required light intensity, and thus minimizing the photothermal damage to living cells, thereby extending cell viability in optical trapping and cell manipulation applications. To this end, a thorough characterization of cell viability in optical trapping environments was performed. This study also demonstrates the technique using spatial light modulation in patterned manipulation of live cell arrays over a broad area.