| The stromal vascular fraction(SVF)is a cell population isolated from the adipose tissue,Now it has been widely used in the field of tissue regeneration.SVF contains hematopoietic stem/progenitor cells,but the number is small and homing ability is low.In the current difficult situation of hematopoietic stem cell donors,how to effectively expand hematopoietic stem/progenitor cells in SVF and improve their homing ability is an important problem that needs to be solved urgently.Adipose derived stromal cells(ADSCs)are pluripotent stem cells that promote hematopoietic reconstitution after transplantation.However,the safety of ADSCs limits its wide application in clinical practice.The exosomes derived from adipose mesenchymal stem cells are membranous vesicles that release ADSCs into the extracellular matrix.Exosomes-mediated paracrine mechanisms play a role in ADSCs-based therapies.AIM:1.To explore the effect of ADSC-exsomes on the proliferation and homing of hematopoietic cells in SVF.2.Provide a theoretical basis for the clinical application of SVF and a new idea for repairing hematopoietic dysfunction.Methods:1.Type? collagenase shaker digests human adipose tissue to isolate SVF.Then the way of immunemagnetic beads was used to sort SVF-CD34 + cells.The hematopoietic function of SVF-CD34 + cells was confirmed by flow cytometry analysis,hematopoietic stem cell colony culture and spleen colony.2.ADSCs were isolated and purified by adherent culture method,and their phenotype and functional identification were identified.ADSCs-exosomes were isolated from the 3rd generation ADSCs by ultrafiltration and density gradient centrifugation.Exosomes were identified by the method of transmission electron microscopy(TEM),the nanoparticle visibility analysis system and the exosomes surface marker protein by Western Blot.3.ADSC-exosomes were co-cultured with SVF-CD34 + cells.The number of cells,CD34 + cells,cell cycle and hematopoietic stem cell colonies were detected at different time points to explore the effect of ADSCs-exosomes on cell proliferation.SVF-CD34 + cells were labeled with CM-DIL and implanted with ADSC-exosomes into Total body irradiated(TBI)NOD/SCID mice.The localization of SVF-CD34 +cells in bone marrow and peripheral organs was observed under fluorescence microscope.Flow cytometry was used to detect the number of fluorescently labeled cells in the bone marrow cavity.Real-time quantitative PCR was used to detect the expression of CXCR4 mRNA in bone marrow mononuclear cells before and after transplantation to investigate the effect of exosomes on homing of SVF-CD34 +cells.4.ADSC-exosomes were co-transplanted with SVF-CD34 + cells.To investigate whether ADSC-exosomes contribute to hematopoietic reconstitution of SVF-CD34 +cells by means of survival analysis,blood routine monitoring,bone marrow routine,GVHD assessment,flow cytometry hCD45 ratio.Result:1.The isolated SVF-CD34 + cells in human adipose tissue haveimmunophenotypic and functional types of hematopoietic stem/progenitor cells.2.Separation of ADSC-exosomes by ultrafiltration Combined with Density Gradient Centrifugation.Transmission electron microscopy shows ADSC-exosmes is a group of round or oval membranous small vesicles with complete membrane.Analysis of nanoparticle visibility showed that the diameter distribution of exosmes was about 76 nm.Exosomes related proteins CD63,CD9,CD81 were positive expression.These results are consistent with the characteristics of exosomes.3.ADSC-exosomes were co-cultured with SVF-CD34 + cells.It was show that ADSC-exosomes have the proliferation function on the SVF-CD34 + cells in a concentration dependent way.Co-cultured for 7 days there are still enough cells in the pre-division,help to maintain its stem cell characteristics.ADSC-exosomes increased the expression of CXCR4 mRNA in SVF-CD34 + cells.In vivo it was confirmed that ADSC-exosomes co-transplantation could make more SVF-CD34 +cells effectively homing to the bone marrow cavity.Real-time quantitative PCR results showed that the transcription level of CXCR4 gene was higher than that before transplantation.4.ADSC-exosomes and SVF-CD34 + cells were co-transplanted into NOD/SCID mice.the recovery time of peripheral blood was shorter than that of group SVF-CD34+ cells.Conclusion:In this study,we successfully established SVF-CD34 + cells from human adipose tissue and confirmed the hematopoietic potential of SVF-CD34 + cells,and successfully isolated and identified ADSC-exosomes.ADSC-exosomes support the proliferation of SVF-CD34 + cells in vitro and contribute to the effective homing of SVF-CD34 + cells and the hematopoietic reconstitution after transplantation.The mechanism may be related to the regulation of SDF-1/CXCR4 signaling pathway.This will provide experimental evidence for the clinical application of SVF in the field of hematopoietic stem cell transplantation,and also provide a new way and solution for the effective amplification of SVF-CD34 + cells in vitro. |
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