Erythropoietin directly remodels the clonal composition of murine hematopoietic multipotent progenitor cells.

The cytokine erythropoietin (EPO) is a potent inducer of erythrocyte development and one of the most prescribed biopharmaceuticals. The action of EPO on erythroid progenitor cells is well established, but its direct action on hematopoietic stem and progenitor cells (HSPCs) is still debated. Here, using cellular barcoding, we traced the differentiation of hundreds of single murine HSPCs, after ex vivo EPO exposure and transplantation, in five different hematopoietic cell lineages, and observed the transient occurrence of high-output myeloid-erythroid-megakaryocyte-biased and myeloid-B-cell-dendritic cell-biased clones. Single-cell RNA sequencing analysis of ex vivo EPO-exposed HSPCs revealed that EPO induced the upregulation of erythroid associated genes in a subset of HSPCs, overlapping with multipotent progenitor (MPP) 1 and MPP2. Transplantation of barcoded EPO-exposed MPP2 confirmed their enrichment in myeloid-erythroid-biased clones. Collectively, our data show that EPO does act directly on MPP independent of the niche and modulates fate by remodeling the clonal composition of the MPP pool.

A Multitubular Kidney-on-Chip to Decipher Pathophysiological Mechanisms in Renal Cystic Diseases.

Autosomal Dominant Polycystic Kidney Disease (ADPKD) is a major renal pathology provoked by the deletion of PKD1 or PKD2 genes leading to local renal tubule dilation followed by the formation of numerous cysts, ending up with renal failure in adulthood. In vivo, renal tubules are tightly packed, so that dilating tubules and expanding cysts may have mechanical influence on adjacent tubules. To decipher the role of this coupling between adjacent tubules, we developed a kidney-on-chip reproducing parallel networks of tightly packed tubes. This original microdevice is composed of cylindrical hollow tubes of physiological dimensions, parallel and closely packed with 100-200 µm spacing, embedded in a collagen I matrix. These multitubular systems were properly colonized by different types of renal cells with long-term survival, up to 2 months. While no significant tube dilation over time was observed with Madin-Darby Canine Kidney (MDCK) cells, wild-type mouse proximal tubule (PCT) cells, or with PCT Pkd1 +/- cells (with only one functional Pkd1 allele), we observed a typical 1.5-fold increase in tube diameter with isogenic PCT Pkd1 -/- cells, an ADPKD cellular model. This tube dilation was associated with an increased cell proliferation, as well as a decrease in F-actin stress fibers density along the tube axis. With this kidney-on-chip model, we also observed that for larger tube spacing, PCT Pkd1 -/- tube deformations were not spatially correlated with adjacent tubes whereas for shorter spacing, tube deformations were increased between adjacent tubes. Our device reveals the interplay between tightly packed renal tubes, constituting a pioneering tool well-adapted to further study kidney pathophysiology.

Engineering small tubes with changes in diameter for the study of kidney cell organization.

Multicellular tubes are structures ubiquitously found during development and in adult organisms. Their topologies (diameter, direction or branching), together with their mechanical characteristics, play fundamental roles in organ function and in the emergence of pathologies. In tubes of micrometric range diameters, typically found in the vascular system, renal tubules or excretory ducts, cells are submitted to a strong curvature and confinement effects in addition to flow. Then, small tubes with change in diameter are submitted to a local gradient of shear stress and curvature, which may lead to complex mechanotransduction responses along tubes, and may be involved in the onset or propagation of cystic or obstructive pathologies. We describe here a simple method to build a microfluidic device that integrates cylindrical channels with changes in diameter that mimic in vivo tube geometries. This microfabrication approach is based on molding of etched tungsten wires, which can achieve on a flexible way any change in diameter in a polydimethylsiloxane (PDMS) microdevice. The interest of this biomimetic multitube system has been evidenced by reproducing renal tubules on chip. In particular, renal cell lines were successfully seeded and grown in PDMS circular tubes with a transition between 80 µm and 50 µm diameters. Thanks to this biomimetic platform, the effect of the tube curvature has been investigated especially regarding cell morphology and orientation. The effect of shear stress on confluent cells has also been assessed simultaneously in both parts of tubes. It is thus possible to study interconnected cell response to differential constraints which is of central importance when mimicking tubes present in the organism.

Gradients of Rac1 Nanoclusters Support Spatial Patterns of Rac1 Signaling.

Rac1 is a small RhoGTPase switch that orchestrates actin branching in space and time and protrusion/retraction cycles of the lamellipodia at the cell front during mesenchymal migration. Biosensor imaging has revealed a graded concentration of active GTP-loaded Rac1 in protruding regions of the cell. Here, using single-molecule imaging and super-resolution microscopy, we show an additional supramolecular organization of Rac1. We find that Rac1 partitions and is immobilized into nanoclusters of 50-100 molecules each. These nanoclusters assemble because of the interaction of the polybasic tail of Rac1 with the phosphoinositide lipids PIP2 and PIP3. The additional interactions with GEFs and possibly GAPs, downstream effectors, and other partners are responsible for an enrichment of Rac1 nanoclusters in protruding regions of the cell. Our results show that subcellular patterns of Rac1 activity are supported by gradients of signaling nanodomains of heterogeneous molecular composition, which presumably act as discrete signaling platforms.