Exploiting hPSCs-derived kidney organoids to study the mechanobiology of nephrogenesis

Abstract

[eng] The isolation and generation of human pluripotent stem cells (hPSCs) has allowed research in human organogenesis to excel, by allowing the reproduction of the fundamental principles of tissue morphogenesis in vitro. Particularly in the field of kidney development, cutting edge research has led to the successful generation of kidney organoids from hPSCs by mimicking in vitro biochemical renal inductive signals that occur during organogenesis. However, tissue formation and morphogenesis don’t solely rely on biochemical and genetic instructions they are also influenced by mechanical signals that drive migration, differentiation, and growth within the forming organ, regulated through local internal forces and dynamics of ECM stiffness and viscosity. While the key morphogens and signalling pathways involved in kidney morphogenesis have been identified, the role of mechanical cues during kidney development and formation of congenital anomalies of the kidneys and urinary tract (CAKUT) has remained elusive. This thesis describes the development of a high throughput system for 2D kidney differentiation (namely RV emergence and nephron-like formation) on soft PDMS substrates which allows the exposure of hPSCs-derived kidney progenitor cells to physical constraints in the form of substrate rigidity and geometry, compatible with traction force measurements. We have validated the system for supporting RV emergence and nephron-like formation in 2D in the background of male ES[4] and female CB40 kidney progenitor cells, as well as measured traction forces exerted by kidney progenitors in wildtype and CAKUT-related phenotypes. We found that wildtype kidney progenitors exert increasing traction forces as differentiation progresses, while in the CAKUT-related backgrounds these forces are diminished. We identified a correlation between extent of kidney differentiation and exposure to increasing curvature of micropattern geometry. In this regard, wildtype progenitors seeded on triangular geometries displayed lower extent of differentiation (as described by immunofluorescence expression) and higher traction forces compared to circle and square counterparts. As such, our experimental pipeline and quantitative analysis have set the bases for further experiments to mechanistically couple how RV emergence and nephron differentiation is driven by changes in the expression of specific renal markers and cell force generation.