Schematic of the KSC epithelial induction protocol.

<p>KSC epithelial differentiation was determined by Transwell co-culture differentiation assay. Hypoxia-injured RTECs and KSCs were prepared and plated in the Transwell inserts and wells, respectively. Cells were co-cultured in induction media for three days, moved to renal epithelial cell growth medium (REGM) for two days, and then changed back to induction medium. This series was repeated twice for a total 10 days. The inserts containing hypoxia-injured RTECs were renewed every three days.</p

Embryonic stem (ES) cell marker expression in KSCs.

<p>(A) <i>Nanog</i>, <i>Oct4/Pou5f1</i>, and <i>Sox2</i> gene expression in KSCs and RTECs. Data represents the mean fold change ± standard error of the mean (SEM) when comparing KSCs to RTECs. All values were normalized to <i>Gapdh</i> expression. (**<i>P</i> < 0.01). B: Western blot analysis of ES markers in KSCs and RTECs. β-actin was used as an internal control.</p

Determination of KSC differentiation abilities.

<p>(A,B) KSC adipogenic and osteogenic differentiation was assessed by (A) oil red O and (B) alizarin red staining, respectively (Magnification: ×200). (C) Epithelial cell differentiation was examined by immunofluorescence staining for the mature epithelial cell markers CK18 (red, ×200), ZO-1 (red, ×200), and E-cadherin (green, ×100). Nuclei are counterstained with DAPI (blue).</p

Hyperglycemic Stress Impairs the Stemness Capacity of Kidney Stem Cells in Rats

<div><p>The incidence of acute kidney injury in patients with diabetes is significantly higher than that of patients without diabetes, and may be associated with the poor stemness capacity of kidney stem cells (KSCs) and limited recovery of injured renal tubules. To investigate the effects of hyperglycemic stress on KSC stemness, KSCs were isolated from the rat renal papilla and analyzed for their self-renewal and differentiation abilities. Our results showed that isolated KSCs expressed the mesenchymal stem cell markers N-cadherin, Nestin, CD133, CD29, CD90, and CD73. Moreover, KSCs co-cultured with hypoxia-injured renal tubular epithelial cell (RTECs) induced the expression of the mature epithelial cell marker CK18, suggesting that the KSCs could differentiate into RTECs in vitro. However, KSC proliferation, differentiation ability and tolerance to hypoxia were decreased in high-glucose cultures. Taken together, these results suggest the high-glucose microenvironment can damage the reparative ability of KSCs. It may result in a decreased of recovery capability of renal tubules from injury.</p></div

Morphological characterization of renal papillary cells.

<p>(A) Primary cell culture on day 3. Cells exhibited colony-like growth and diverse morphology. (B) Primary cell culture on day 5. Epithelioid and fibroblast-like morphologies were observed. (C,D) P2 cells after passage on day 5. (E,F) P3 cells showed a short fusiform or dendritic shape, and epithelioid cells had almost completely disappeared. (G,H) P5 cells exhibited a spindle-shape. (Magnification: C, E, G, ×40; A, B, F, H, ×100; D, ×200).</p

KSC growth curves after culturing in normal-glucose or high-glucose medium.

<p>Growth curves for cells cultured in high-glucose or normal-glucose media were graphed based on the OD values at 450 nm. (*<i>P</i> < 0.05, **<i>P</i> < 0.01).</p

Effects of high glucose on KSC differentiation into epithelial cells.

<p>(A) Flow cytometry analysis of CK18 expression (<i>P</i> = 0.045); (B) E-cadherin and AQP-1 gene expression analysis by qRT-PCR. (*<i>P</i> < 0.05, <i>**P</i> < 0.01).</p

Phenotypic characterization of renal papillary cells.

<p>(A) Immunofluorescence analysis for markers of activated fibroblasts (α-SMA, green; Vimentin, red), mesenchymal stem cells (N-cadherin, green), and epithelial cells (CK18, red; E-cadherin, green; ZO-1, red). Nuclei were counterstained with DAPI (blue) (Magnification: ×100). (B) Immunofluorescence staining for the stem cell expression makers Nestin (green) and CD133 (red). Nuclei are stained with DAPI (blue). (Magnification: ×200). (C) Flow cytometry analysis for the MSC markers CD29 (i), CD90 (ii), and CD73 (iii), and the hematopoietic stem cell marker CD45 (iv).</p

Additional file 1 of Establishment of human hematopoietic organoids for evaluation of hematopoietic injury and regeneration effect

Additional file 1: Supplemental methods

Additional file 1: of Embryonic stem cell preconditioned microenvironment suppresses tumorigenic properties in breast cancer

Supplementary information contains Table S1 presenting primer sequences used for real-time PCR, Figure S1 showing mES cell characterization and reporter gene cells used in the experiment, Figure S2 showing decreased proliferation of 4T1 cells by direct cell–cell contact of ES cells, Figure S3 showing ES-CM inhibited Stat3 signaling pathway in 4T1 cells (related to Figure 3), and Figure S4 showing ES-CM inhibit tumor growth in vivo (related to Figure 5). (DOCX 4638 kb