Stem cells derived from human amniotic fluid contribute to acute kidney injury recovery

Abstract

Stem cells isolated from human amniotic fluid are gaining attention with regard to their therapeutic potential. In this work, we investigated whether these cells contribute to tubular regeneration after experimental acute kidney injury. Cells expressing stem cell markers with multidifferentiative potential were isolated from human amniotic fluid. The regenerative potential of human amniotic fluid stem cells was compared with that of bone marrow-derived human mesenchymal stem cells. We found that the intravenous injection of 3.5 × 10(5) human amniotic fluid stem cells into nonimmune-competent mice with glycerol-induced acute kidney injury was followed by rapid normalization of renal function compared with injection of mesenchymal stem cells. Both stem cell types showed enhanced tubular cell proliferation and reduced apoptosis. Mesenchymal stem cells were more efficient in inducing proliferation than amniotic fluid-derived stem cells, which, in contrast, were more antiapoptotic. Both cell types were found to accumulate within the peritubular capillaries and the interstitium, but amniotic fluid stem cells were more persistent than mesenchymal stem cells. In vitro experiments demonstrated that the two cell types produced different cytokines and growth factors, suggesting that a combination of different mediators is involved in their biological actions. These results suggest that the amniotic fluid-derived stem cells may improve renal regeneration in acute kidney injury, but they are not more effective than mesenchymal stem cells.

Figure 1

Experimental schedule. Schematic representation of the protocol of glycerol-induced AKI and treatment with either hAFSCs or MSCs. Glycerol was injected intramuscularly at time 0. The arrow at day three indicates the administration of 350,000 cells or vehicle alone; the subsequent arrows indicate the time of sacrifice.

Figure 2

Fluorescence-activated cell sorter analysis of hAFSCs. Representative fluorescence-activated cell sorter analyses for the expression of surface markers CD44, CD45, CD73, CD90, CD 105, CD166, and SSEA4 and the intracellular transcription factor Oct4. The gray line indicates the isotypic controls. All analyzed hAFSCs (n = 12) showed similar results in passages 2 to 5. PE, phycoerythrin; FITC, fluorescein isothiocyanate.

Figure 3

Differentiation characteristics of hAFSCs. A: Representative of osteogenic differentiation detected as calcium oxalate deposits detectable by Van Kossa staining. B: Representative of adipogenic differentiation visualized by Oil Red O staining of the intracellular lipid vesicles. C: Representative of chondrogenic differentiation detected by Alcian Blue staining. Micrographs are representative of 12 experiments.

Figure 4

Evaluation of renal function. Changes in renal function were measured by BUN (A) and creatinine (B). Glycerol injection resulted in elevated BUN levels starting at day three. In animals injected with hAFSCs or MSCs, BUN normalized 48 hours after injection and remained close to baseline throughout the experiment (A). Similar results were obtained measuring creatinine levels (B). Analysis of variance was performed: *P < 0.05, stem cells treated versus untreated.

Figure 5

Histology of AKI in hAFSC- or MSC-treated and control untreated mice. A and B: Representative micrographs of renal tissue from mice on day 3 (A) and day 5 (B) after glycerol injection, showing tubular necrosis, tubular protein casts, and loss of brush border; vacuolization of the epithelial cells of the proximal tubules is visible in the high power images. C and D: Representative micrographs of renal tissue from mice on day 5 after glycerol injection treated with hAFSCs (C) or MSCs (D), showing signs of recovery of tissue injury. E–G: Representative micrographs of renal tissue from mice on day 8 after glycerol injection, showing the persistence of renal injury in untreated animals (E) and signs of recovery in hAFSC-treated (F) or MSC-treated (G) animals. H–J: Representative micrographs of renal tissue from mice on day 21 showing the normal morphology of tissue in animals untreated (H) or treated with hAFSCs (I) and MSCs (J). Original magnification of each panel: ×200 (left); ×400 (right).

Figure 6

Tubular proliferation in AKI untreated or treated with hAFSCs or MSCs. A and B: Graphs illustrating the quantification of BrdU (A)- and Pcna (B)-positive cells at days 5, 8, and 21 in AKI mice treated with hAFSCs or MSCs or injected with saline as a control. Statistical significance was calculated using analysis of variance with the Newman-Keuls multicomparison test: *P < 0.05, stem cells in AKI-treated versus untreated mice; **P < 0.005, MSCs versus hAFSCs. C: Representative micrographs showing BrdU- and Pcna-positive cells in kidney tissue from AKI mice injected with saline or hAFSCs at day eight. Original magnification, ×400. Ctrl, control.

Figure 7

Detection of hAFSCs or MSCs within the kidneys of AKI mice by CFSE or HLA immunostaining. A and B: Graphs illustrating the quantification of CFSE-labeled cells (A) and of HLA-positive cells (B). Student's t-test was performed between hAFSC- and MSC-treated AKI mice at each time point. *P < 0.001. C: Representative confocal micrographs showing the presence of MSCs or hAFSCs within the kidneys of mice with AKI at days 4, 8, and 21. Tubular epithelial cells were stained for cytokeratin (red). The CFSE-labeled and HLA-positive cells were detected as green fluorescence. Nuclei were counterstained with 4,6-diamidino-2-phenylindole (blue).D: Representative confocal micrographs showing the absence of detection of CFSE-labeled or HLA-positive cells within the kidneys of control mice (Ctrl). Original magnification, ×400.

Figure 8

Cytokines/growth factors released into the conditioned medium of hAFSCs and MSCs and effects on tubular cell proliferation. A: Evaluation of cytokines/growth factors released into the conditioned medium of 1 × 10⁶ hAFSCs and MSCs after a 12-hour incubation in RPMI 1640 plus 0.5% bovine serum albumin. Cytokines were measured using a multiplex cytokine array. Data represent the mean ± SD of five different cell lines. FGF, fibroblast growth factor; PDGF, platelet-derived growth factor; VEGF, vascular endothelial growth factor; LIF, leukemia inhibitory factor; NGF, nerve growth factor; SCF, stem cell factor; SDF, stromal derived factor; HGF, hepatocyte growth factor. B: Proliferation of TECs induced by the conditioned medium (CM) of hAFSCs and MSCs was evaluated by incorporation of BrdU and expressed as the percent increase over unstimulated control cells (Ctrl). The role of LIF in proliferation induced by the conditioned medium was evaluated using 3 μg/ml anti-LIF blocking antibodies (Ab-LIF). Data represent the mean ± SD of three different experiments performed in duplicate. Analysis of variance with Dunnett's comparison test: *P < 0.005, CM plus Ab-LIF versus CM alone.