Understanding the spatial organization of gene expression with single nucleotide resolution requires localizing the sequences of expressed RNA transcripts within a cell in situ. Here we describe fluorescent in situ RNA sequencing (FISSEQ), in which stably cross-linked cDNA amplicons are sequenced within a biological sample. Using 30-base reads from 8,742 genes in situ, we examined RNA expression and localization in human primary fibroblasts using a simulated wound healing assay. FISSEQ is compatible with tissue sections and whole mount embryos, and reduces the limitations of optical resolution and noisy signals on single molecule detection. Our platform enables massively parallel detection of genetic elements, including gene transcripts and molecular barcodes, and can be used to investigate cellular phenotype, gene regulation, and environment in situ.

______________________________________________________________________Development of new therapeutics for chronic respiratory diseases, such as asthma and chronic obstructive pulmonary disease (COPD), which pose a huge public health burden 1 , have been hindered by the inability to study organ-level complexities of lung inflammation in vitro. While hospitalization and mortality due to these diseases are often the consequences of exacerbations triggered by pathogens 2, 3 , there is currently no way to study these processes in human lung outside of the clinical setting. Animal models of asthma and COPD exist; however, their clinical relevance is questionable because the anatomy, immune system and inflammatory responses exhibited by animal lungs differ greatly from those in humans [4][5][6] . For example, mucin-producing cells, which are central to the development of asthma, are less frequent in the respiratory tree of mice and rats compared with humans 6 . Neutrophils that increase dramatically in the lungs of patients with COPD and severe asthma 7-9 also comprise only 10-25% of circulating leukocytes in mice, whereas they represent 50-70% in humans 5 . Because many animal models fail to predict drug activities in humans, the pharmaceutical and biotechnology industries strive to reduce or replace animal models for drug testing whenever possible 10 .Airway inflammatory diseases have been modeled in vitro using cultures of primary or immortalized human epithelial cells, sometimes positioned at an air-liquid interface to induce epithelial differentiation 11 or using co-cultures of airway epithelium and tissue-resident immune cells (e.g., macrophages or dendritic cells) 12 . However, lung inflammation is mediated by organ-level responses that involve complex tissuetissue interactions between the lung airway epithelium and underlying microvascular endothelium that modulate immune reactions to respiratory pathogens and allergens [13][14][15] and alter the vascular cell adhesion molecular machinery that recruits circulating immune cells, such as neutrophils. This is important because neutrophil accumulation in the lung is associated with enhanced severity of airflow limitation in COPD patients 7 and it plays a critical role in severe asthma as well 8 . Unfortunately, it is not possible to study complex interactions among airway epithelium, endothelium and circulating neutrophils using existing in vitro lung models because most fail to recapitulate normal functional coupling between the epithelium and endothelium, and none enable analysis of recruitment of circulating immune cells under active fluid flow. This latter point is crucial because neutrophil adhesion to inflamed endothelium involves initial rolling along the luminal surface of endothelium mediated by E-selectin, which is then followed by firm adhesion to and this dynamic shear stress-dependent response cannot be studied in a physiologically relevant way using static cell cultures.Advances in microsystems engineering have recently made it possible to create bio...

Kidney organoids derived from human pluripotent stem cells exhibit glomerular- and tubular-like compartments that are largely avascular and immature in static culture. Here, we report an in vitro method for culturing kidney organoids under flow on millifluidic chips, which greatly expands their endogenous pool of endothelial progenitor cells (EPCs) and generates vascular networks with perfusable lumens surrounded by mural cells. Vascularized kidney organoids cultured under flow exhibit more mature podocyte and tubular compartments with enhanced cellular polarity and adult gene expression, compared to static controls. However, the association of vessels with these compartments is reduced upon disrupting the endogenous VEGF gradient. Glomerular vascular development progresses through intermediate stages akin to the embryonic mammalian kidney’s formation of capillary loops abutting foot processes. The ability to induce substantial vascularization and morphological maturation of kidney organoids in vitro under flow opens new avenues for studying kidney development, disease, and regeneration.

An in vitro model of the human kidney glomerulus — the major site of blood filtration — could facilitate drug discovery and illuminate kidney-disease mechanisms. Microfluidic organ-on-a-chip technology has been used to model the human proximal tubule, yet a kidney-glomerulus-on-a-chip has not been possible because of the lack of functional human podocytes — the cells that regulate selective permeability in the glomerulus. Here, we demonstrate an efficient (> 90%) and chemically defined method for directing the differentiation of human induced pluripotent stem (hiPS) cells into podocytes that express markers of the mature phenotype (nephrin+, WT1+, podocin+, Pax2−) and that exhibit primary and secondary foot processes. We also show that the hiPS-cell-derived podocytes produce glomerular basement-membrane collagen and recapitulate the natural tissue/tissue interface of the glomerulus, as well as the differential clearance of albumin and inulin, when co-cultured with human glomerular endothelial cells in an organ-on-a-chip microfluidic device. The glomerulus-on-a-chip also mimics adriamycin-induced albuminuria and podocyte injury. This in vitro model of human glomerular function with mature human podocytes may facilitate drug development and personalized-medicine applications.