22 research outputs found
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Generation of kidney tubular organoids from human pluripotent stem cells
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
Selective depletion of mouse kidney proximal straight tubule cells causes acute kidney injury
The proximal straight tubule (S3 segment) of the kidney is highly susceptible to ischemia and toxic insults but has a remarkable capacity to repair its structure and function. In response to such injuries, complex processes take place to regenerate the epithelial cells of the S3 segment; however, the precise molecular mechanisms of this regeneration are still being investigated. By applying the “toxin receptor mediated cell knockout” method under the control of the S3 segment-specific promoter/enhancer, Gsl5, which drives core 2 β-1,6-N-acetylglucosaminyltransferase gene expression, we established a transgenic mouse line expressing the human diphtheria toxin (DT) receptor only in the S3 segment. The administration of DT to these transgenic mice caused the selective ablation of S3 segment cells in a dose-dependent manner, and transgenic mice exhibited polyuria containing serum albumin and subsequently developed oliguria. An increase in the concentration of blood urea nitrogen was also observed, and the peak BUN levels occurred 3–7 days after DT administration. Histological analysis revealed that the most severe injury occurred in the S3 segments of the proximal tubule, in which tubular cells were exfoliated into the tubular lumen. In addition, aquaporin 7, which is localized exclusively to the S3 segment, was diminished. These results indicate that this transgenic mouse can suffer acute kidney injury (AKI) caused by S3 segment-specific damage after DT administration. This transgenic line offers an excellent model to uncover the mechanisms of AKI and its rapid recovery
An investigation of mechanism of progression of kidney disease using renal tubular cells differentiated from ES/iPS cells
Mechanism of regeneration of renal tubular cells and differentiation from ES and iPS cells to renal tubular cells
Localization of inositol 1,4,5-trisphosphate receptors in the rat kidney
Localization of inositol 1,4,5-trisphosphate receptors in the rat kidney. Inositol 1,4,5-trisphosphate (IP3) receptors (IP3Rs) serve as intracellular calcium release channels involved in signal transduction of various hormones in the kidney. Molecular cloning studies have shown that there are three types of IP3R, designated type 1, type 2, and type 3. To characterize their localizations in the rat kidney, we employed immunohistochemical studies using type-specific monoclonal antibodies that were raised against the 15 C-terminal amino acids of each type of IP3R. Type 1 was detected in glomerular mesangial cells and vascular smooth muscle cells. Type 2 was expressed exclusively in intercalated cells of collecting ducts from the cortex to the inner medulla. Type 3 was expressed in vascular smooth muscle cells, glomerular mesangial cells, and some cells of cortical collecting ducts, probably principal cells. As to the subcellular distribution, type 1 and type 2 showed a homogenous distribution in the cytoplasm, whereas type 3 was present mainly in the basolateral portion of the cytoplasm. These results indicate that IP3R isoforms were expressed in a cell-specific manner. The heterogeneous subcellular localizations among the IP3R types suggests compartmentalization of distinct IP3-sensitive Ca2+ pools
KSP-positive cells formed tubular structures in Matrigel.
<p>(a) Light microscopy of KSP-positive and KSP-negative cells under the conditions indicated in the upper left corner of the pictures on the day after flow cytometry. A micron scale is shown at the lower right corner of the pictures. (b) Electron microscopy of a cross section of the tubular structures formed by KSP-positive cells. The asterisks indicate the lumens, the arrowheads point to microvilli-like structures, and an arrow indicates a tight junction. Magnification is shown at the lower right corner.</p
Wnt4 enhanced the tubular formation of KSP-positive cells.
<p>(a) Fluorescence microscopy of tubular structures formed by KSP-positive cells derived from CAG-GFP EB3. The upper lane shows pictures of tubular structures of KSP-positive cells co-cultured with NIH3T3-Wnt4, and the lower lane shows those co-cultured with NIH3T3-Mock. Scale bar, 200 µm. (b) Bar graph indicating the numbers of tubular structures under the indicated conditions. One tubular structure from a branching to a branching is counted as one. (c) Immunofluorescence of KSP-positive or KSP-negative cells showing KSP and human-specific mitochondria as a negative control. Scale bar, 400 µm. (d) PCR showing the expression of Osr1, KSP and GAPDH of KSP-positive and -negative cells after three days of 3D culture in Matrigel with NIH3T3-Wnt4.</p