3日間GSK3β阻害剤を用いて, hPSCを中胚葉細胞に分化させ, 次に, これらの細胞を腎上皮増殖培地で培養し, KSP+細胞を誘導した。抗KSP抗体によるフローサイトメトリーによりKSP+細胞を純化すると, それらの細胞は腎尿細管細胞のあらゆるセグメントの特徴を示し, またKSP+細胞を3Dマトリゲルで培養するとin vitroで尿細管オルガノイドを形成した。KSP+細胞による尿細管オルガノイドの形成は機能的な腎尿細管の獲得を誘導した。また, KSP+細胞は, マウス胚性腎臓細胞とともに培養すると, 3D尿細管構造内で自己組み立てを行うので, ヒトとマウスのキメラ腎臓培養物の作製が可能であった。
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.
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