Mouse Embryonic Stem Cell–Derived Embryoid Bodies Generate Progenitors That Integrate Long Term into Renal Proximal Tubules In Vivo

Abstract

The metanephric kidney is a mesodermal organ that develops as a result of reciprocal interactions between the ureteric bud and the blastema. The generation of embryonic stem (ES) cell-derived progenitors offers potential for regenerative therapies but is often limited by development of tumor formation. Because brachyury (T) denotes mesoderm specification, a mouse ES cell line with green fluorescence protein (GFP) knocked into the functional T locus as well as lacZ in the ROSA26 locus (LacZ/T/GFP) was used in cell selection and lineage tracing. In the absence of leukemia inhibitory factor, mouse ES cells give rise to embryoid bodies that can differentiate into mesoderm. Culture conditions were optimized (4 d, 10 ng/ml Activin-A) to generate maximal numbers of renal progenitor populations identified by expression of the specific combination of renal markers cadherin-11, WT-1, Pax-2, and Wnt-4. LacZ/T/GFP؉ cells were further enriched by FACS selection. Five days after injection of LacZ/T/GFP؉ cells into embryonic kidney explants in organ culture, ␤-galactosidase immunohistochemistry showed incorporation into blastemal cells of the nephrogenic zone. After a single injection into developing live newborn mouse kidneys, co-localization studies showed that the LacZ/T/GFP؉ cells were stably integrated into proximal tubules with normal morphology and normal polarization of alkaline phosphatase and aquaporin-1 for 7 mo, without teratoma formation. It is concluded that defined differentiation of ES cells into embryoid bodies with Activin-A and selection for T expression provides a means to isolate and purify renal proximal tubular progenitor cells with the potential for safe use in regenerative therapies.

Embryonic Stem Cells Proliferate and Differentiate when Seeded into Kidney Scaffolds

“…46 In addition, yet another study showed renal proximal tubular progenitor cells can be differentiated from ES cells using activin-A and sorted for Brachyury (T). 47 These methods can be used to differentiate the cells before scaffold seeding, providing a more kidney-specific lineage, or integrated into the culture system, allowing the cells to form ECM-mediated structures as they grow and differentiate. Other additives that may be used to enhance the outcome and shape organ development include hyaluronic acid, which stimulates tubule differentiation, or other growth factors such as vascular endothelial growth factor to support endothelialization of the vasculature.…”

In vitro organogenesis from undifferentiated cells in Xenopus

“…This induction method established for the animal cap assay has been applied to other cell induction experiments. Recently, it has been reported that ES cells treated with activin and RA differentiate into nephric tissues (Kim and Dressler, 2005;Vigneau et al, 2007).…”

Stem cell options for kidney disease

“…Furthermore, when injected into metanephric explants culture, primed EBs showed 100% incorporation into developing renal tubules. Similarly, murine ES cells primed in vitro with activin-A alone or BMP-4 differentiate into cells expressing markers of the intermediate mesoderm, early kidney development-as well as renal tubule-specific markers [60,61]. A recent study has shown that human amniotic-fluid derived stem cells are capable of forming early nephron structures (renal vesicles, S-and comma-shaped bodies) when injected into embryonic mouse kidneys.…”