Isolation and culture of cells
Human placentas were obtained from caesarean sections from healthy women with informed consent. The amnion layer was separated from chorion layer and washed several times with PBS and cut into pieces of 1.0cm2 and digested with 0.25% trypsin at 37°C for 30 min. After that, amniotic epithelial cells were removed gently. And then, the amnion was digested again under the same condition. Finally, the amnion was washed by PBS and cut into 1mm2 and cultured in α-MEM (1×; Gibco, USA, C12571) supplemented with 10% FBS (Gibco, Brazil, A31608-02). All the hAMSCs used in experiments were third-passage to fifth-passage. Suspension of P3 hAMSCs were counted and adjusted into 1×106 cells/mL.
Identification of human amniotic mesenchymal stem cells
For cell identification, cells were incubated with mouse anti-human CD90-PE antibody (BD Pharmingen™, catalog number: 561970), mouse anti-human CD105-PE antibody (eBioscience, catalog number: 12-1057-42), mouse anti-human CD73-PE antibody (BD Pharmingen™, catalog number: 561014), mouse anti-human CD45 -PE antibody (BD Pharmingen™, catalog number: 560975), mouse anti-human CD11b-PE-Cy™7antibody (BD Pharmingen™, catalog number: 561098), mouse anti-human CD34-PE antibody (BD Pharmingen™,catalog number: 560941) mouse and anti-human HLA-DR-PE antibody (BD Pharmingen™, catalog number: 560943) at 4℃ for 30min. Cells were washed by PBS and adjusted into 400μL and then analyzed by flow cytometry.
Adipogenic and osteogenic differentiation
To analyze adipogenic and osteogenic differentiation, passage 3 hAMSCs were seeded at a density of 1.5×105 cells/well in a six-well plate. When the cells reached 100% confluence, cells were incubated in human mesenchymal stem cell adipogenic differentiation medium (Pythonbio, catalog number: 20191008), for 21 days, the adipogenic induction medium was replaced every 3 days. Oil red O (Solarbio, catalog number: G1260) staining was performed to assess the differentiation potential. For osteogenic differentiation, hAMSCs were cultured with human mesenchymal stem cell osteogenic differentiation medium (Pythonbio, catalog number: 20191009) for 21 days, induction medium was replaced every 3 days. The differentiation potential for osteogenesis was assessed by Alizarin Red (pH 4.2, 40 mM) (Solarbio, catalog number: G1452) staining.
NPG mice 8-10 weeks of age were obtained from Beijing Vitalstar Biotechnology Co.,Ltd (laboratory animal production license No.SCXK2016-0167). Animals were housed in a specific pathogen-free facility in microisolator cages, given autoclaved food and maintained on acidified autoclaved water and a solution of gentamicin. All animal experiments were approved by the laboratory animal ethics review committee of southern medical university. All animal procedures were in accordance with the National Institute about laboratory animal care and use.
Human peripheral blood mononuclear cells collection
Human PBMC were collected from healthy volunteers. Each donor singed written informed consent. PBMC were collected in sodium citrate and purified by Ficoll-Hypaque (Solarbio) density centrifugation and washed in phosphate-buffered saline (PBS). Then suspended in red blood cell lysis buffer (Solarbio) at 4℃ for 15min and washed again in PBS. And finally suspended in PBS for injection into NPG mice from tail vein.
Induction of xenogeneic aGVHD in NPG mice
All mice were given 200 cGy irradiation 3-4h before cell injection unless indicated otherwise. Then were injected with 3×106 of PBMC suspended into 500μL PBS intravenously. During all experiments, each mouse was graded according xeno-aGVHD clinical scoring system (Table 1). The symptoms of xeno-GVHD including weight loss, hunched posture, ruffled fur, reduced mobility and diarrhea. Mice survived till the 28 day endpoint and those which suffered from severe xeno-GVHD (weight loss＞25%, severe hunched posture, severe ruffled fur, less or no mobility or hematochezia) were euthanized.
GFP-labelled hAMSCs and tracing hAMSC in vivo
hAMSCs were planted to 24-well plate, cell concentration was 3×105/mL, 500μL per well. Then the 24-well plate was placed in an incubator condition of 37℃ and 5% CO2. 24 hours later, culture media was disposed, 250μL culture media and 1.6μL GFP-pseudovirion (Hanbio) were added, the rest of the 250μL culture media was added 4 hours later and then placed in the incubator. After 24 hours, culture media was disposed, 500μL culture media was added and then placed in the incubator. GFP labeled hAMSCs could be found by fluorescence microscope in two to three days. The suspension of GFP labeled hAMSCs was adjusted into 5×105 cells/mL and injected into NPG mice via tail vein. These mice were euthanized after 24h and 72h respectively, blood and tissues such as liver, spleen, lung and gut were collected. Single cell suspensions were obtained by grinding liver, spleen, lung, gut. Finally, single cell suspensions of tissues and blood were analyzed by flow cytometry to detected GFP labeled signal. For immunofluorescence, target organs were fixed with 4% paraformaldehyde for 24 hours and then dehydrated with 30% sucrose. After more than 48 hours target organs were embedded in a frozen slicer at -25°C and made into frozen slices. Recipient cells were distinguished by DAPI counterstaining.
Tissues were collected at the time of necropsy, fixed in 10% buffered formalin, and embedded in paraffin. For routine histology, sections were stained with hematoxylin and eosin. For immunohistochemistry, sections were heated at 60°C for 20 min, then deparaffinized and hydrated with xylene and alcohol baths for staining. The slides were heated in 0.01M citrate-buffer solution (pH 6.0) for 10 min in a microwave oven, then cooled down to room temperature. Immunohistochemical staining was performed with mAbs specific for human CD45 (Abcam, Massachusetts, US). For immunofluorescence, target organs were fixed with 4% paraformaldehyde for 24 hours and then dehydrated with 30% sucrose. After more than 48 hours target organs were embedded in a frozen slicer at -25°C and made into frozen slices. Recipient cells were distinguished by DAPI counterstaining.
Flow Cytometric Analysis
Mouse anti-human CD3-PE-Cyanine7 antibody (eBioscience, catalog number: 25-0038-42), mouse anti-human CD4-FITC antibody (BD Pharmingen™, Catalog Number:340133), mouse anti-human CD8-PE (BD Pharmingen™, Catalog Number:340046), mouse anti-human CD25-APC antibody (Biolegend, Catalog Number: 302610), mouse anti-human Foxp3-PE(eBioscience Catalog Number: 12-4777-42). Peripheral blood, livers, spleens, lungs and guts were collected at the time of necropsy and analyzed by flow cytometry. Single cell suspensions were obtained by grinding liver, spleen, lung, gut and blood sample was processed with red blood cell lysis buffer (Solarbio) at 4℃ according to the protocol. All samples were stained with antibodies or isotype matched control IgG for 30min at 4℃ in the dark. Then analyzed on a BD FACS CantoII with FlowJo 10.0.
Analysis of the cytokines by cytometric bead array (CBA)
Cytometric bead array (CBA) human Th1/Th2/Th17 cytokine kit (BD, Catalog Number: 560484).
Peripheral blood and single cell suspensions of each target organ were centrifugate in a condition of 4℃, 3000rpm/min and 20min. Mixing human Th1/Th2/Th17 cytokine capture beads, vortex the mixed Capture Beads and add 50 µL to all assay tubes, add 50 µL of each sample to the appropriately labeled sample tubes and add 50 µL of the Human Th1/Th2/Th17 PE detection reagent to all assay tubes. Incubate the assay tubes for 3 hours at room temperature, protected from light. add 1 mL of Wash Buffer to each assay tube and centrifuge at 200g for 5 minutes, carefully aspirate and discard the supernatant from each assay tube. add 300 µL of Wash Buffer to each assay tube to resuspend the bead pellet. Acquire the samples on the flow cytometer (BD FACS cantoII), analyze Human Th1/Th2/Th17 Cytokine data using FCAP Array software.
Comparisons between two means used the independent samples t-test. Comparisons of three or more means used one-way ANOVA. Survival curves were made with the Kaplan–Meier method. P＜0.05 were considered as significant, and for all figures, *p<0.05, **p<0.01, ***p<0.001.
All statistical analyses were performed with GraphPad Prism 8.0.