Abstract
Recent advances in stem cell research have resulted in methods to generate kidney organoids from human pluripotent stem cells (hPSCs), which contain cells of multiple lineages including nephron epithelial cells. Methods to purify specific types of cells from differentiated hPSCs, however, have not been established well. For bioengineering, cell transplantation, and disease modeling, it would be useful to establish those methods to obtain pure populations of specific types of kidney cells. Here, we report a simple two-step differentiation protocol to generate kidney tubular organoids from hPSCs with direct purification of KSP (kidney specific protein)-positive cells using anti-KSP antibody. We first differentiated hPSCs into mesoderm cells using a glycogen synthase kinase-3β inhibitor for 3 days, then cultured cells in renal epithelial growth medium to induce KSP+ cells. We purified KSP+ cells using flow cytometry with anti-KSP antibody, which exhibited characteristics of all segments of kidney tubular cells and cultured KSP+ cells in 3D Matrigel, which formed tubular organoids in vitro. The formation of tubular organoids by KSP+ cells induced the acquisition of functional kidney tubules. KSP+ cells also allowed for the generation of chimeric kidney cultures in which human cells self-assembled into 3D tubular structures in combination with mouse embryonic kidney cells.
Highlights
To obtain pure population of kidney tubular cells, we focused on one of cadherins, Kidney-specific protein (KSP; cadherin 16) which is exclusively expressed in kidney lineage cells including ureteric buds, developing nephrons, mesonephric tubules, Bowman’s capsules, proximal tubules, loops of Henle, and distal and collecting tubules[26]
To determine whether additional growth factors to renal epithelial growth medium (REGM) further enhance KSP expression, we tested activin A (10 ng/ml), hepatocyte growth factor (HGF; 10 ng/ml) or insulin-like growth factor 1 (IGF-1; 10 ng/ml) from day 3 to day 10 of differentiation, since our previous study showed that HGF and IGF-1 increased KSP expression in mouse ESCs differentiated with DMEM supplemented with activin A 10 ng/ml[13]
Any of activing A, HGF or IGF-1, did not further increase KSP expression compared to REGM alone on day 10 of differentiation (Fig. 1c), suggesting that REGM itself was optimized for differentiation of human embryonic stem cells (hESCs) into kidney tubular cells
Summary
To obtain pure population of kidney tubular cells, we focused on one of cadherins, Kidney-specific protein (KSP; cadherin 16) which is exclusively expressed in kidney lineage cells including ureteric buds, developing nephrons, mesonephric tubules, Bowman’s capsules, proximal tubules, loops of Henle, and distal and collecting tubules[26]. We previously generated a monoclonal antibody against the extracellular domain of KSP, which enabled to purify KSP+cells by flow cytometry[13]. Building on these prior reports, the aim of this study was to establish a rapid and simple differentiation protocol of human embryonic stem cells (hESCs) into kidney tubular cells with purification of KSP+cells and to generate tubular organoids in 3D culture systems. We demonstrated that the KSP+cells form tubular structures in chimeric culture with dissociated mouse metanephric kidneys, supporting our hypothesis that purification of KSP+cells from differentiated hPSCs provides pure population of kidney tubular cells
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