Abstract
ObjectivesKidney disease is emerging as a critical medical problem worldwide. Because of limited treatment options for the damaged kidney, stem cell treatment is becoming an alternative therapeutic approach. Of many possible human stem cell sources, pluripotent stem cells are most attractive due to their self-renewal and pluripotent capacity. However, little is known about the derivation of renal lineage cells from human pluripotent stem cells (hPSCs). In this study, we developed a novel protocol for differentiation of nephron progenitor cells (NPCs) from hPSCs in a serum- and feeder-free system.Materials and MethodsWe designed step-wise protocols for differentiation of human pluripotent stem cells toward primitive streak, intermediate mesoderm and NPCs by recapitulating normal nephrogenesis. Expression of key marker genes was examined by RT-PCR, real time RT-PCR and immunocytochemistry. Each experiment was independently performed three times to confirm its reproducibility.ResultsAfter modification of culture period and concentration of exogenous factors, hPSCs can differentiate into NPCs that markedly express specific marker genes such as SIX2, GDNF, HOXD11, WT1 and CITED1 in addition to OSR1, PAX2, SALL1 and EYA1. Moreover, NPCs possess the potential of bidirectional differentiation into both renal tubular epithelial cells and glomerular podocytes in defined culture conditions. In particular, approximately 70% of SYN-positive cells were obtained from hPSC-derived NPCs after podocytes induction. NPCs can also form in vitro tubule-like structures in three dimensional culture systems.ConclusionsOur novel protocol for hPSCs differentiation into NPCs can be useful for producing alternative sources of cell replacement therapy and disease modeling for human kidney diseases.
Highlights
The kidneys have crucial regulatory roles in the body, including control of fluid-electrolyte, acid-base and blood pressure, and the excretion of waste products
After modification of culture period and concentration of exogenous factors, human pluripotent stem cells (hPSCs) can differentiate into nephron progenitor cells (NPCs) that markedly express specific marker genes such as SIX2, GDNF, HOXD11, WT1 and CITED1 in addition to OSR1, PAX2, SALL1 and EYA1
Approximately 70% of SYN-positive cells were obtained from hPSC-derived NPCs after podocytes induction
Summary
The kidneys have crucial regulatory roles in the body, including control of fluid-electrolyte, acid-base and blood pressure, and the excretion of waste products. Based on the normal renal developmental steps, metanephros is the final step of nephrogenesis during gestation [12,13,14] It consists of two structures derived from intermediate mesoderm, the ureteric bud (UB) and metanephric mesenchyme (MM) [14,15]. A robust induction of intermediate mesoderm (IM) cells from hPSCs was accomplished with phenotypic characterization [32] These cells were not kidney progenitor cells which have more therapeutic potential in vivo [21,24]. Ureteric bud-committed kidney progenitor-like cells was recently derived from hPSCs in vitro and in vivo [33]. We differentiated hPSCs into NPCs by step-wise approach in a serum- and feeder-free condition These NPCs were able to turn into two cell types within nephrons, renal tubular epithelial-cells and glomerular podocytes in vitro
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