Direct Isolation and Characterization of Human Nephron Progenitors

Abstract

Mature nephrons originate from a small population of uninduced nephrogenic progenitor cells (NPs) within the cap mesenchyme. These cells are characterized by the coexpression of SIX2 and CITED1. Many studies on mouse models as well as on human pluripotent stem cells have advanced our knowledge of NPs, but very little is known about this population in humans, since it is exhausted before birth and strategies for its direct isolation are still limited. Here we report an efficient protocol for direct isolation of human NPs without genetic manipulation or stepwise induction procedures. With the use of RNA‐labeling probes, we isolated SIX2+CITED1+ cells from human fetal kidney for the first time. We confirmed their nephrogenic state by gene profiling and evaluated their nephrogenic capabilities in giving rise to mature renal cells. We also evaluated the ability to culture these cells without complete loss of SIX2 and CITED1 expression over time. In addition to defining the gene profile of human NPs, this in vitro system facilitates studies of human renal development and provides a novel tool for renal regeneration and bioengineering purposes. Stem Cells Translational Medicine 2017;6:419–433

Reading First Coordinates from the Nephrogenic Zone in Human Fetal Kidney

“…So far it is known, the special operation is under the control of protocadherin (cadherin family member14: FAT4/dachsous cadherin related1: Dchs1) signaling [62]. As a result, epithelial stem cells in the tip of a CD ampulla meet GDNF + /Six2 + /CITED1 + mesenchymal cells [51, 52]. At the same time, a reciprocal exchange of morphogens within the niche takes place [37, 38].…”

“…In zone 4, only 2 layers of mesenchymal stem cells are present. Due to their close vicinity to the tip of a CD ampulla, the inner mesenchymal cell layer corresponds to GDNF + /Six2 + /CITED1 + cells [51, 52]. It is particularly important that these cells send out projections to establish functional contacts with neighboring epithelial cells via tunneling nanotubes [58].…”

“…Moreover, it has been known for years that morphogenetic interactions between epithelial stem cells in the tip of a CD ampulla and juxtaposed nephrogenic mesenchymal (GDNF + /Six2 + /CITED1 + ) cells lead to induction and initial formation of a nephron [37, 38, 51, 52]. It is obvious that the micrographs and morphometric data shown here are still sparse, that further parameters must be considered and the stock of measurements must be expanded (Table 1).…”

“…Occasionally, a multilayered cap mesenchyme becomes visible, when sections are analyzed that lie oblique to the axis of related CD tubules. Finally, the interface between the mesenchymal stem cell layer (GDNF + /Six2 + /CITED1 + ) juxtaposed to the tip of a CD ampulla containing epithelial stem cells defines the center of an individual stem cell niche enabling induction and initial formation of an individual nephron [51, 52, 54-56]. …”

“…Overall, the contained components represent the cell biological setting to maintain stemness, branching morphogenesis, induction, and initial formation of individual nephrons. In this context, one must consider that all of the different tasks are mastered by a set of morphogens and nephron progenitor cells [51, 52]. …”