ABSTRACT
A major cause of chronic kidney disease (CKD) is glomerular disease, which can be attributed to a spectrum of podocyte disorders. Podocytes are non-proliferative, terminally differentiated cells. Thus, the limited supply of primary podocytes impedes CKD research. Differentiation of human pluripotent stem cells (hPSCs) into podocytes has the potential to produce podocytes for disease modeling, drug screening, and cell therapies. In the podocyte differentiation process described here, hPSCs are first induced to primitive streak-like cells by activating canonical Wnt signaling. Next, these cells progress to mesoderm precursors, proliferative nephron progenitors, and eventually become mature podocytes by culturing in a serum-free medium. Podocytes generated via this protocol adopt podocyte morphology, express canonical podocyte markers, and exhibit podocyte phenotypes, including albumin uptake and TGF-ß1 triggered cell death. This study provides a simple, defined strategy to generate podocytes for in vitro modeling of podocyte development and disease or for cell therapies.
Subject(s)
Cell Differentiation , Pluripotent Stem Cells/cytology , Podocytes/cytology , Cells, Cultured , Humans , Mesoderm/cytology , Mesoderm/metabolism , Nanog Homeobox Protein/genetics , Nanog Homeobox Protein/metabolism , Octamer Transcription Factor-3/genetics , Octamer Transcription Factor-3/metabolism , Phenotype , Pluripotent Stem Cells/metabolism , Podocytes/metabolism , Primitive Streak/cytology , Primitive Streak/metabolism , Wnt Proteins/metabolismABSTRACT
The blood-brain barrier (BBB) is composed of specialized endothelial cells that are critical to neurological health. A key tool for understanding human BBB development and its role in neurological disease is a reliable and scalable source of functional brain microvascular endothelial cells (BMECs). Human pluripotent stem cells (hPSCs) can theoretically generate unlimited quantities of any cell lineage in vitro, including BMECs, for disease modeling, drug screening, and cell-based therapies. We demonstrate a facile, chemically defined method to differentiate hPSCs to BMECs in a developmentally relevant progression via small-molecule activation of key signaling pathways. hPSCs are first induced to mesoderm commitment by activating canonical Wnt signaling. Next, these mesoderm precursors progress to endothelial progenitors, and treatment with retinoic acid leads to acquisition of BBB-specific markers and phenotypes. hPSC-derived BMECs generated via this protocol exhibit endothelial properties, including tube formation and low-density lipoprotein uptake, as well as efflux transporter activities characteristic of BMECs. Notably, these cells exhibit high transendothelial electrical resistance above 3000 ohm·cm2. These hPSC-derived BMECs serve as a robust human in vitro BBB model that can be used to study brain disease and inform therapeutic development.
