SYM-07: Stem Cells and Organs-on-Chips


Justin Cooper-White1,2,3, Nick Glass2, Minoru Takasato4, Pei Xuan Er4, Ernst Wolvetang1, Melissa Little4, Drew Titmarsh1

1Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, Australia;
2CSIRO Manufacturing Flagship;
3The University of Queensland, Australia;
4Murdoch Children’s Research Institute, Australia

The differentiation of human pluripotent stem cells (hPSCs) towards kidney is of great interest for developmental biology, toxicology and regenerative medicine. Herein, directed differentiation into kidney cell populations and organoids have been explored using a microfluidic system and image cytometry in a systematic and quantitative manner. We have investigated this complex differentiation process under soluble factor perturbation using a full factorial 3 by 3 microbioreactor array (MBA) previously developed in the Cooper-White lab. This allowed for 27 unique soluble factor combination inputs. These inputs are then perfused through 10 serial wells allowing for the investigation of potential paracrine and autocrine signalling, generating 270 unique conditions within the MBA. Cells were differentiated towards intermediate mesoderm cell populations (PAX2+), before running several different factors across the MBA. Initial experiments examined the effect of known signalling pathways on the differentiation of hPSCs towards renal cell populations at day 9 and day 12 time points of the differentiation protocol. Results confirmed the essential factors for the differentiation towards kidney like cell phenotypes and the condensation of ureteric epithelial cells (GATA3+ECAD+). This effect was synergistically enhanced in the presence of some of the factors investigated but also gave rise to stromal/mesangial cell populations of renal origin (GATA3+ECAD-). Early nephrons (WT1+ECAD+) and metanephric mesenchyme (WT1+ECAD-) have also been observed to form around the ureteric epithelial structures. Image cytometry was then performed across the entire device to provide FACs-like data for the population within each well. Further parameters based on clustering structures of cells were also investigated and quantified with respect to soluble factor and paracrine signalling. Microfluidic-enabled factorial optimisation of directed differentiation into complex structures of the human kidney was confirmed. Important cell subtypes of the kidney were identified and quantified in many different conditions, providing novel insight into developmental pathways and kidney organogensis.

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