Stem cells from human amniotic fluid exert immunoregulatory function via secreted indoleamine 2,3-dioxygenase1

Abstract

Although human amniotic fluid does contain different populations of foetal-derived stem cells, scanty information is available on the stemness and the potential immunomodulatory activity of in vitro expanded, amniotic fluid stem cells. By means of a methodology unrequiring immune selection, we isolated and characterized different stem cell types from second-trimester human amniotic fluid samples (human amniotic fluid stem cells, HASCs). Of those populations, one was characterized by a fast doubling time, and cells were thus designated as fHASCs. Cells maintained their original phenotype under prolonged in vitro passaging, and they were able to originate embryoid bodies. Moreover, fHASCs exhibited regulatory properties when treated with interferon (IFN)-γ, including induction of the immunomodulatory enzyme indoleamine 2,3-dioxygenase 1 (IDO1). On coculture with human peripheral blood mononuclear cells, IFN-γ-treated fHASCs caused significantly decreased T-cell proliferation and increased frequency in CD4(+) CD25(+) FOXP3(+) regulatory T cells. Both effects required an intact IDO1 function and were cell contact-independent. An unprecedented finding in our study was that purified vesicles from IFN-γ-treated fHASCs abundantly expressed the functional IDO1 protein, and those vesicles were endowed with an fHASC-like regulatory function. In vivo, fHASCs were capable of immunoregulatory function, promoting allograft survival in a mouse model of allogeneic skin transplantation. This was concurrent with the expansion of CD4(+) CD25(+) Foxp3(+) T cells in graft-draining lymph nodes from recipient mice. Thus fHASCs, or vesicles thereof, may represent a novel opportunity for immunoregulatory maneuvers both in vitro and in vivo.

Keywords: T cells; cell culture; cloning; immunosuppression; lymphocytes; pluripotent stem cells.

Figure 1

fHASC morphology and shape of the colonies. (A) fHASCs with fibroblast-like morphology; (B) colony shape is similar to dermatoglyphics (inset). Representative data of one from five different lines.

Figure 2

Stem cell-like phenotype of fHASCs. (A) Analysis of expression of pluripotent markers by real-time PCR in fHASCs. Data (mean ± SD of three experiments. This is explained a few lines below, prior to (B)) are presented as normalized transcript expression in the samples relative to normalized transcript expression in control cultures (that is, a pool of eight aliquots of amniotic fluid cells, in which fold change = 1; dotted line). iPSCs, derived as described in Material and methods, were used as a positive control. *P < 0.05; **P < 0.001 (Shapiro test). (B) Analysis of pluripotent marker expression by FACS in fHASCs. (C and D) Analysis of the embryonic stem cell markers SSEA-3 and SSEA-4 by flow cytometry in fHASCs. Data are representative of three independent experiments conducted on five fHASC lines.

Figure 3

Differentiation potential HASCs. (A) Representative pictures of embryoid bodies (EBs) formed by HASCs in hanging-drop cultures. fHASC-derived EBs that were capable of sustained growth in vitro (T0, 4 days; T10, 10 days; T20, 20 days). (B) EBs stained with haematoxylin/eosin. EB images were obtained by phase-contrast microscopy (×20 magnification). (C) Marker expression before and after EB formation as assessed by RT-PCR (normalized to β-actin). (D) Representative pictures of fHASC differentiation into adipocytes and osteocytes respectively. Differentiation was monitored with Sudan III staining of lipid droplets in terminal adipocyte differentiation and von Kossa staining of calcium deposition in the extracellular matrix in terminal osteoblast differentiation; nuclear staining was with Mayer’s haematoxylin. (E) qRT-PCR analysis of mRNA levels of specific adipogenic (PPARG, LPL, FABP4) and osteogenic (ALPL, OCN, OPN, RUNX2) markers for fHASCs). Analysis was performed at day 21 of differentiation as compared to day 0, which corresponds to fold change = 1. All data shown are representative of three independent experiments conducted on five fHASC lines (*P < 0.05).

Figure 4

fHASCs express immunoregulatory IDO1. (A) IDO1 detection by immunofluorescence staining in fHASCs that were treated or not with 1000 U/ml IFN-γ for 24 hrs. Nuclei were counterstained with DAPI (blue) and IDO1 was revealed using a fluorescently labelled secondary antibody. Data are representative of one of three independent experiments using five fHASC lines. (B) Western blot analysis of IDO1 protein expression in fHASCs treated or not with 100, 200, 500 or 1000 U/ml IFN-γ for 24 hrs; β-tubulin was used as a control. (C) IDO1 enzymatic activity was measured by HPLC quantification of tryptophan conversion into kynurenine. Data are representative of three independent experiments involving five fHASC lines ***P<0.05–0.01.

Figure 5

Treg induction by fHASCs requires IDO1 but not cell–cell contact. (A) Regulatory T-cell enumeration by cytofluorimetric analysis, presented as the proportion of CD4⁺ CD25⁺ FOXP3⁺ cells in PBMCs after 96-hr coculture with fHASCs that had been treated or not with IFN-γ, or transfected with IDO1 siRNA (IDO1 siRNA) or control siRNA (c siRNA). (B) Representative plots from one of three experiments. (C and D) Panels refer to the same experimental conditions as in (A) and (B) but with the cocultured PBMCs and fHASCs being separated by transwell inserts. Data shown in A and C are representative of three independent experiments involving five fHASC lines (**P < 0.01; *P < 0.05).

Figure 6

IFN-γ-stimulated fHASCs release nanovesicles that contain active IDO1 protein. (A) SEM images of fHASCs captured at different magnifications and showing vesicle budding from the plasma membrane. Nanovesicles (NVs) were purified from fHASCs, treated or not with IFN-γ and were visualized by SEM (B). (C) Size distribution of NVs obtained by statistical analysis of SEM images (630 random counts) manipulated using ImageJ software. (D) Hydrodynamic size distribution by DLS of NVs purified from fHASCs. (E) FACS analysis of CFSE-labelled fHASC-derived vesicles. One representative experiment of three. (F) NVs were purified from fHASCs, treated or not with IFN-γ and were subjected to lysis and analysis by Western blot to determine protein content. (G) Kynurenine production by fHASC-derived NVs or intact fHASCs that had been pre-treated with IFN-γ for 24 hrs prior to analysis. (H) Cytoplasmic vesicle-free fraction (CVF) was purified from fHASCs, treated or not with IFN-γ and was analysed by Western blot for IDO1 protein content. One representative experiment of three. (I) Cell proliferation was measured by cytofluorimetric analysis of CD4⁺ EdU⁺ cells in PBMCs after coculture with either fHASC-derived NVs or intact fHASCs that had been treated with IFN-γ for 24 hrs and/or transfected with IDO1 siRNA (IDO1 siRNA) or control siRNA (c siRNA). (J) Regulatory T-cell enumeration by cytofluorimetric analysis of CD4⁺ CD25⁺ Foxp3⁺ cells after 96-hr coculture. Shown are mean values ± SD from three independent experiments involving five fHASC lines (*P < 0.05).

Figure 7

fHASCs promote skin allograft engraftment. (A) Experimental protocol for fHASC-dependent skin allograft tolerance induction. BALB/c tail skin grafts were used as donor material while C57BL/6 were transplant recipients. For tolerance induction, C57BL/6 mice were treated intraperitoneally with a single dose of fHASCs (2 × 10⁶ cells) on day −1. Skin transplantation was performed on day 0. (B) Post-transplantation survival curves in syngeneic skin graft only (C57BL/6 /C57BL/6), black line (n = 10); in skin allograft only (BALB/c /C57BL/6/vehicle), red line (n = 10); and in skin allograft with fHASC treatment (BALB/c /C57BL/6/fHASCs), green line (n = 10). Observation period, 20 days. Log-rank test (*P < 0.05). (C) Representative skin graft appearance and histological evaluation. (I) Complete graft rejection with the scar from vehicle-treated mice; (II) completely healed skin graft without evidence of rejection from fHASC-treated recipients. Haematoxylin and eosin staining of skin graft sections from vehicle (III) or fHASC (IV) recipients. (D) Frequency of CD4⁺ CD25⁺ Foxp3⁺ Treg cells in spleens and graft-draining lymph nodes from treated recipients. Data are mean values ± SD of three independent experiments (n = 9). (E) Representative plots from one of three experiments. (F) TGF-β and IL-10 production from purified CD4⁺ cells from graft-draining lymph nodes, in treated recipients. Data are mean values ± SD of three independent experiments (n = 9).