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Selective In Vitro Propagation of Nephron Progenitors Derived from Embryos and Pluripotent Stem Cells.

ABSTRACT: Nephron progenitors in the embryonic kidney propagate while generating differentiated nephrons. However, in mice, the progenitors terminally differentiate shortly after birth. Here, we report a method for selectively expanding nephron progenitors in vitro in an undifferentiated state. Combinatorial and concentration-dependent stimulation with LIF, FGF2/9, BMP7, and a WNT agonist is critical for expansion. The purified progenitors proliferated beyond the physiological limits observed in vivo, both for cell numbers and lifespan. Neonatal progenitors were maintained for a week, while progenitors from embryonic day 11.5 expanded 1,800-fold for nearly 20 days and still reconstituted 3D nephrons containing glomeruli and renal tubules. Furthermore, progenitors generated from mouse embryonic stem cells and human induced pluripotent cells could be expanded with retained nephron-forming potential. Thus, we have established in vitro conditions for promoting the propagation of nephron progenitors, which will be essential for dissecting the mechanisms of kidney organogenesis and for regenerative medicine.

SUBMITTER: Tanigawa S

PROVIDER: S-EPMC7815166 | biostudies-literature | 2016 Apr

REPOSITORIES: biostudies-literature

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Selective In Vitro Propagation of Nephron Progenitors Derived from Embryos and Pluripotent Stem Cells.

Tanigawa Shunsuke S Taguchi Atsuhiro A Sharma Nirmala N Perantoni Alan O AO Nishinakamura Ryuichi R

Cell reports 20160414 4

Nephron progenitors in the embryonic kidney propagate while generating differentiated nephrons. However, in mice, the progenitors terminally differentiate shortly after birth. Here, we report a method for selectively expanding nephron progenitors in vitro in an undifferentiated state. Combinatorial and concentration-dependent stimulation with LIF, FGF2/9, BMP7, and a WNT agonist is critical for expansion. The purified progenitors proliferated beyond the physiological limits observed in vivo, bot ...[more]

PMID: 27149838

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Project description:BackgroundHuman induced pluripotent stem cells (hiPSCs) offer a renewable source of cells for the generation of hematopoietic cells for cell-based therapy, disease modeling, and drug screening. However, current serum/feeder-free differentiation protocols rely on the use of various cytokines, which makes the process very costly or the generation of embryoid bodies (EBs), which are labor-intensive and can cause heterogeneity during differentiation. Here, we report a simple feeder and serum-free monolayer protocol for efficient generation of iPSC-derived multipotent hematoendothelial progenitors (HEPs), which can further differentiate into endothelial and hematopoietic cells including erythroid and T lineages.MethodsFormation of HEPs from iPSCs was initiated by inhibition of GSK3 signaling for 2 days followed by the addition of VEGF and FGF2 for 3 days. The HEPs were further induced toward mature endothelial cells (ECs) in an angiogenic condition and toward T cells by co-culturing with OP9-DL1 feeder cells. Endothelial-to-hematopoietic transition (EHT) of the HEPs was further promoted by supplementation with the TGF-β signaling inhibitor. Erythroid differentiation was performed by culturing the hematopoietic stem/progenitor cells (HSPCs) in a three-stage erythroid liquid culture system.ResultsOur protocol significantly enhanced the number of KDR+ CD34+ CD31+ HEPs on day 5 of differentiation. Further culture of HEPs in angiogenic conditions promoted the formation of mature ECs, which expressed CD34, CD31, CD144, vWF, and ICAM-1, and could exhibit the formation of vascular-like network and acetylated low-density lipoprotein (Ac-LDL) uptake. In addition, the HEPs were differentiated into CD8+ T lymphocytes, which could be expanded up to 34-fold upon TCR stimulation. Inhibition of TGF-β signaling at the HEP stage promoted EHT and yielded a large number of HSPCs expressing CD34 and CD43. Upon erythroid differentiation, these HSPCs were expanded up to 40-fold and displayed morphological changes following stages of erythroid development.ConclusionThis protocol offers an efficient and simple approach for the generation of multipotent HEPs and could be adapted to generate desired blood cells in large numbers for applications in basic research including developmental study, disease modeling, and drug screening as well as in regenerative medicine.

Dataset Information

Selective In Vitro Propagation of Nephron Progenitors Derived from Embryos and Pluripotent Stem Cells.

Publications

Selective In Vitro Propagation of Nephron Progenitors Derived from Embryos and Pluripotent Stem Cells.

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