FC041: Regeneration of Nephrons and Renal Stroma using the Renal Developmental Environment of Animal Fetuses
Authors: Saito Yatsumu , Yamanaka Shuichiro , Matsumoto Naoto , Takamura Tsuyoshi , Fujimoto Toshinari , Matsui Kenji , Tajiri Susumu , Matsumoto Kei , Yokoo Takashi 
Published: May 2022

Abstract

BACKGROUND AND AIMS
The kidney is derived from three types of progenitor cells: nephron progenitor cells (NPCs), stromal progenitor cells (SPCs) and ureteric buds (UBs). Extensive research in regenerative medicine has made it possible to induce pluripotent stem cells (PSCs) to develop into NPCs and UBs in vitro and generate the organoids of nephrons and collecting ducts. Renal stroma, including mesangial cells and erythropoietin- and renin-producing cells, is essential for maintaining biological functions. However, to our knowledge, the induction of SPC differentiation from PSCs has not been established yet. Moreover, the renal stroma cannot mature in the in vitro environment, making it difficult to generate the whole kidney, including the renal stroma. In this study, we attempted to regenerate the renal stroma in the in vivo environment by transplanting rat SPCs into the nephrogenic zone of mouse fetuses to provide rat SPCs with the renal developmental mechanism of heterologous mouse fetuses. Furthermore, we developed an interspecies dual replacement of progenitor (i-DROP) system wherein two progenitor cell lines, NPCs and SPCs, were genetically modified and replaced with transplanted progenitor cells and examined the feasibility of the regeneration of kidneys, including nephrons and renal stroma.

METHOD
We crossed Foxd1GC/+ mice with R26RDTR/DTR mice to generate Foxd1GC/+; R26RDTR/+ mice (Foxd–DTR mice) can eliminate Foxd1-positive SPCs. A group of SPC-rich cells extracted from green fluorescent protein (GFP) rat metanephros targeting platelet-derived growth factor receptor alpha (PDGFRa) was transplanted into the kidneys of Foxd1–DTR mouse fetuses. Subsequently, the organ-cultured metanephros or the transplanted metanephros in immunodeficient NOD/Shi- scid IL2rγnull (NOG) mice or Sprague–Dawley (SD) rats were evaluated using immunostaining. Next, we assessed whether the i-DROP system could regenerate interspecies nephrons and stroma. We crossed Six2TGC/+; Foxd1GC/+ mice obtained from Six2TGC/+ mice and Foxd1GC/+ mice with R26RDTR/DTR mice to generate Six2TGC/+; Foxd1GC/+; and R26RDTR/+ mice (Six2/Foxd1–DTR mice) that can simultaneously eliminate Six2-positive NPCs and Foxd1-positive SPCs. The SPCs targeted to PDGFRa+ and NPCs targeted to PDGFRa−/integrin alpha 8 (Itga8)+ from GFP rat metanephros were simultaneously transferred to Six2/Foxd1–DTR mouse metanephros and evaluated similarly.

RESULTS
In the organ culture of Foxd1–DTR mouse metanephros, SPCs around the cap mesenchyme replaced from mouse to GFP rat. We achieved extensive rat stromal cell regeneration in mouse kidneys, including endocrine functions such as erythropoietin production in the rat in response to anemia in the host mouse. Next, in the organ culture of the hind kidney of Six2/Foxd1–DTR mice equipped with the i-DROP system, the NPCs and SPCs of GFP rats aggregated around the mouse UB and the cap mesenchyme reconstituted. In vivo, rat nephrons and renal stroma were regenerated simultaneously in the mouse kidneys. The newly generated glomeruli were composed of GFP-positive rat cells, both podocytes and mesangial cells of different lineages (Figure 1). Intravenous injection of fluorescently labeled dextran into host mice revealed that the regenerated nephrons had glomerular filtration and tubular reabsorption capabilities. Furthermore, taking the xenotransplantation of mouse metanephros into SD rats as a control, the transplantation of regenerated rat kidneys into SD rats using our method resulted in reduced xenogenic immune rejection.

CONCLUSION
The newly developed i-DROP system regenerated multifunctional kidneys, composed of nephrons and renal stroma originating from different species, with urine production and endocrine functions. Functional human kidneys could be regenerated in the future using the renal developmental mechanism of large heterologous animals, such as pigs.