Empirical Study On Adipose Tissue-derived Mesenchymal Stem Cells In Severe Aplastic Anemia

Background and ObjectMesenchymal stem cells (MSCs) can be isolated from various adult tissues of human by their ability of adhering plastic culture plate wall. MSCs can proliferate extensively in vitro, and differentiate under appropriate conditions in bone, cartilage, and other mesenchymal tissues but also into multiple other cells derived from the germ layers including neural cells. Human MSCs have been recently proposed for the treatment of various diseases, including acute graft-versus-host disease (GVHD), elevate the engraftment of HSCs in co-transplantation with hematopoietic stem cells (HSCs), neural system disorder, acute myocardial infarction.The first non-hematopoietic mesenchymal stem cells (MSCs) were discovered by Friedenstein in 1976. Toksoz thought these cells are also stromal cells, structural components of the bone marrow that support ex vivo culture of hematopoiesis by providing extracellular matrix components, cytokines, and growth factors. The maintenance of hematopoiesis is believed to be regulated and controlled by the BM microenvironment. The non-hematopoietic connective tissue elements provide a system not only of structural support for developing hematopoietic cells but also of functional support for the process of hematopoiesis through the provision of a unique microenvironment that provides appropriate growth regulatory molecules and promotes cell-cell and cell-extracellular matrix (ECM) interactions (REFS). Bone marrow mesenchymal stem cells produce IL-6,IL-11,SCF,flt-3 ligand and other hematopoietic active cytokines, that can improve LTC-IC expansion and promote the differentiation of CD34⁺ cells. These studies suggest mesenchymal stem cells are capable of supporting hematopoietic and accelerating hematopoietic recovery.Severe Aplastic anemia is a rare hematopoietic stem cell disorder that results in the decreasing of peripheral blood cells and bone marrow cells. Now, bone marrow transplantation has already become the unique effective therapeutic instrument for severe aplastic anemia. But, hematopoietic stem cell transplantation also faces much differenty such as hematopoietic recovery slowly, that have puzzled many investigators who are engaged on stem cells. In addition, recent publications in xenogeneic bone marrow MSCs transplantation models that were injected via tail vein have shown that MSCs enhances the hematopoietic recovery effectively in xenogeneic recipients. Our study is just based on this to explore the hematopoietic reconstitution potential of mice adipose tissue-derived mesenchymal stem cells. So we carried out transplantation experimentation in vivo in order to provide rationale for clinical application.Materials and Methods1. Experimental animals and regentsAll normal animals used were 6-8-week-old female C57BL/6 (H-2K^b)mice and male BALB/c (H-2K^d) obtained from the experimental animal center of zhengzhou university. DMEM,MCDB-201,F12,Gln-glutamine,insulin,epidermal growth factor; fetal bovine serum,horse serum ; collagenase; pamcreatin; antibody: H-2K^d,fluorescein isothiocyanate(FITC)-conjugated Secondary Antibody.2. Ex vivo male BALB/c mice adipose tissue-derived MSCs cultureMice were killed by cervical dislocation and abdominal subcutaneous fatty tissue isolated. To remove the hematopoietic cells by D-Hanks, the fatty tissue was digested two hours by collagenase, then, the mononuclear cells were prepared by gradient centrifugation at 900g for 5 minutes. Then cells were washed, counted, plated at 25/cm² onto culture flasks in58%DMEM/F12 +40%MCDB-201 supplemented with 2% fetal bovine serum,1×Iusulin-Transferrin-Selenium,1×linoleic acid-bovine serum albumin and incubated in 5% CO2 at 37℃. Medium was replaced, and the non adherent cells were removed at 48 hours of initial culture and every 3 or 4 days thereafter. When 70% to 80% confluent, adherent cells were trypsinized (0.25% trypsin at 37℃for 5 minutes), harvested, and split 1:3.3. Flow cytometry and Differentiation assaysThe adipose tissue-derived adherent cells were determined the antigen expression profile by flow cytometry. To induce osteogenic differentiation cultures were daily fed with MSC medium to which was added 10 mMβ-glycerophosphate, 50mg/ml ascorbic acid and 10^-9 M dexamethasonfor up to 3 weeks. Mineralization of the extracellular matrix was visualized by von kossa staining. Adipogenic differentiation was induced by passaging cells at a 1:10 dilution in MSC medium + 5μg/ml insulin and 10^-9M dexamethasone. Adipogenic differentiation was visualized by Oil Red O Method for Lipofuchsin.4. Prepared for mice bone marrow single cell suspensionsC57BL/6 mice were killed by cervical dislocation and femora and tibiae isolated. After dissection of attached muscle and connective tissue from the bones, the marrow was extruded by clipping of the epiphysial ends of the bones and flushing using D-Hank's prechilled. Single cell suspensions were prepared from the marrow plugs by blow repeatly. To remove the hematopoietic cells by EDTA-NH₄Cland to wash two times by D-Hank's, the cell suspensions were detected cell viability by trypan blue exclusion.5. The mice model of severe aplastic anemiaThe model of aplastic anemia was formed by ⁶⁰Co-γ+CH+CY. Specific steps as follow: the model of aplastic anemia was formed by injecting 50.0mg.kg^-1 cyclophoph- amide and 62.5 mg.kg^-1 chloramohenicol for three days on the fourth day after the irradiation with 3.0Gy⁶⁰Co-γ. Mice generate severe aplastic anemia at the eighth days.6. Transplantation in vivoAplastic anemia mice were classified into 3 groups at random. 10days after irradiation, 2×10⁷expanded bone marrow single cells resuspended in PBS were injected into mice together with or without 3×10⁶ adipose tissue-derived MSCs. Control groups included mice receiving only normal sodium. After infusion, bone marrow was collected by flushing the single femur. BMNC (bone marrow nucleated cell) count and CFU-GM yields were observed dynamically.7. H-2K^d detected by flow cytometryBone marrow single cells suspension of transplanted mice Cells were incubated with anti-H-2K^d for overnight and then washed two times by PBS supplemented with 0.01% sodium azide and 0.1%BSA. Cells were incubated with fluorescein isothiocyanate (FITC)-conjugated Secondary Antibody for 45 minutes at 4℃. Cells were washed, resuspended in before-mentioned PBS and analyzed with a FACScan cytometer.8. Reverse transcriptase-polymerase chain reactionOligonucleotide primers of Y chromosome were SRY as follows: forward primer 5'-CT-GCTGTGAACAGACACTAC-3'and reverse primer 5'-GACTCCTCTGACTT CACT-TG-3'. Amplification for Sry was performed under the cycling conditions (3min at 95℃, 30s at 94℃, 50s at 61℃, 40s at 72℃, 8min at 72℃, 28 cycles).9. Statistical analysisStatistical analyses were performed using table curve software (SPSS,Paris, France). Statistical comparisons were made using t-test comparison with two samples mean. P-values of less than 0.05 were considered statistically significa.Results1. The immunophenotype of BALB/c mice adipose tissue-derived MSCsMSCs were defined as cells positive for CD29,CD44,CD105,Flk-1 and negative for CD31,CD34,HLA-DR using FACS analysis.2. Multilineage differentiation of MSCsAdipose tissue-derived MSCs induced by osteogenic differentiation and adipogenic differentiation cultures were visualized mineralization of the extracellular matrix and intracellular lipid droplets respectively using von kossa staining and Oil Red O Method. 3. Re-establish hematopoiesis experiment in vivoThe granulocytic cells count of peripheral blood and femoral bone marrow and the count of CFU-GM by the second week in the experimental group were much higher than those of controls. Y-chromosome-specific PCR and H-2K^d to detect the distribution of donor-derived cells in male mice adipose tissue-derived cells-transplanted mice.ConclusionAdipose tissue-derived MSCs can markly re-establish hematopoiesis and enhancement hematopoiesis recovery in SCT.