Biological Characteristics In Vitro Of Bone Marrow Derived Mesenchymal Stem Cells From Different Resources And The Use Of Human Bone Marrow Derived Mesenchymal Stem Cells In Hematological Stem Cell Transplantation

PART ONE Biological Characteristics in Vitro of Culture Expanded Human Bone Marrow Derived Mesenchymal Stem Cells and Cotransplantation of them with Hematopoietic Stem Cells in Patients with Hematological DiseasesObjective:Bone marrow derived MSCs were expanded and its biological characteristics in vitro were identified.We hope to explore the feasibility and safety of cotransplantation culture-expanded MSCs and peripheral blood stem cells(PBSCs)in patients with hematologic diseases.Methods:Bone marrow mononuclear cells from 16 healthy donors and 4 patients with hematological malignancies after chemotherapy were cultured and expanded ex-vivo.Immunophenotype,adipogenic and osteogenic differentiation potential,karyotype,senescence associatedβ- galactosidase staining and immunosuppressive property of the harvested MSCs were characterized.Five patients received transplantation of culture-expanded MSCs and autologous PBSCs. Fifteen patients were cotransplanted with PBSCs and culture-expanded MSCs from the same donor(12 HLA-identical sibling donors and 3 HLA- haploidentical related donors).Hematopoietic reconstruction,complications and clinical outcomes after transplantation in these patients were observed.Results:1.(1.82±0.38)×10⁶/kg (donor's weight)MSCs were successfully expanded from 25.6±5.1ml bone marrow samples from 16 healthy donors after 3～4 passaging.They were CD73,CD90,CD105 positive and CD34,CD45,CD38,CD10,CD20,CD33,HLA-DR negative.The number of MSCs in the population was more than 95%.They had normal karyotype. They had adipogenic and osteogenic differentiation potential and could inhibit mixed lymphocyte reactions(MLRs).Most cells showed no activity ofβ-galactosidase.2. (1.93±0.57)×10⁶/kg BM-MSCs were harvested from 4 patients with hematological malignancies after 3～4 passaging.They showed the same characteristics as from healthy donors.3.No adverse response about infusion of autologous or allogenic MSCs was observed for five patients undergoing autologous PBSCT.They had rapid hematopoietic reconstructions and without severe complications.They were alive and had been followed for 45～70mo.4.Twelve patients were undergoing HLA-identical sibling cotransplantation.No adverse response was observed during and after the infusion of MSCs.Hematopoietic reconstructions were successful and all had full donor-type chimerism 1 month after transplantation.Two patients(16.67%)developed gradeⅡ～Ⅳacute GVHD.Two patients(16.67%)developed extensive chronic GVHD.Four patients suffered from cytomegalovirus(CMV)infection but were cured at last.Till now,seven patients have been alive for 29～57 months and five patients died.5.MSCs together with HSCs transplantation from the same HLA-haploidentical donor were used in 3 patients without adverse response of infusion.Hematopoietic reconstruction was successful in all and they had full donor-type chimerism 1 month after transplantation.None had radeⅡ～ⅣaGVHD until two patients received donor lymphocyte infusion(DLI)to treat(or prevent)the relapse.They developed had gradeâ…£aGVHD and extensive cGVHD.The other one only had gradeâ… aGVHD and limited cGVHD.Two patients have been followed up for 53 and 43 months respectively,one patient died.Conclusion:MSCs identified by immunophenotype analysis and bi-lineages differentiation assays can be isolated from human bone marrow,expanded effectively by culture.Their had high purity,normal karotype, almost no senescence,are suitable for clinical use.It is safe and feasible to cotransplant patients with culture-expanded MSCs in autologous,HLA-identical sibling and HLA-haploidentical related PBSCT.More samples and analysis in pairs will be needed in the future in order to study the impact of MSCs cotransplanted during HSCT on hematopoietic reconstruction,immunological reconstruction and GVHD. PART TWO Isolation and Culture of Bone Marrow Derived Mesenchymal Stem Cells in Patients with Polycythemia Vera and the Identification of Their in-vitro Biological CharacteristicsObjective:To isolate and culture bone marrow mesenchymal stem cell(MSCs)from polycythemia vera(PV)patients and identify their in-vitro biological characteristics.Methods:Bone marrow was extracted from 9 patients with PV and 6 normal individuals.MSCs were isolated by density gradient centrifugation.The adherent cells were cultured and passaged.The cultured cells harvested from the third passage were assessed.The morphology of the cells was observed by Wright's staining and the growth curve of the cells was plotted by cell counting.The ultrastructure of MSCs was observed with electron microscopy.The cell cycle and immunophenotype of the expanded MSCs were detected by flow cytometry(FCM). Different agents were used to induce MSCs to differentiate into osteocyte and adipocyte.Von Kossa staining and oil-red staining were used to examine the differentiation.Conventional cytogenetics(CC)was carried out to detect karyotype. Alleles specific polymerase chain reaction(AS-PCR)was used to detect JAK2V617F mutation.RT-PCR were used to analyze the expression of multiple hematopoietic growth factors at mRNA level for MSCs.Results:MSCs from patients and normal individuals displayed a fibroblast-like morphology adhering to the culture plate and the doubling time of expand MSC was about 43h.More than 90%cells were at G0/G1 phase and less than 10%cells at S phase of cell cycle.The cells were CD73,CD90 positive,and CD34,CD45,CD133,HLA-DR negative. Under suitable conditions,these MSCs could differentiate into osteocytes and adipocytes.MSCs from patients and normal individuals showed normal karyotype. And JAK2V617F mutation was negative in both MSCs.The same expression pattern was seen in patients and normal individuals that they expressed mRNA of SCF, Flt3-ligand,TPO,LIF,IL-6,IL-11 and did not express G-CSF,GM-CSF and IL-3.Conclusion:MSCs from patients with PV are similar to that from normal individuals.PART THREE In Vitro Aging of Rat Mesenchymal Stem Cells During in-vitro PassagingObjectives:Mesenchymal stem cells(MSCs)are now used in repair medicine and transplantation because of its multipotency and immunomodulatory effect.They need in vitro expansion to get adequate number for clinical use.Human MSCs had been observed to enter senescence during in vitro culture.We evaluated whether the same phenomenon existed during long term culture of rat MSCs.Methods:Bone marrow from 20 male Sprague-Dawley(SD)rats(198±2.12g)were cultured and nonadherent cells were removed three days later.MSCs were identified by osteogenic differentiation and adipocytic differentiation.Cells of each passage were detected for morphology observed by phase contrast microscopy and Wright's staining,ultrastructure by scanning electron microscope,growth curve by CCK-8 kits detection,osteogenic differentiation and von kossa staining,adipocytic differentiation and oil red staining,senescence associatedβ-galactosidase staining, quantitative assay of p16^INK4agene.Results:The cells derived from rat bone marrow were fusiform shaped or polygon,had osteogenic and adipogenic capacity. These showed they were MSCs.Cells were becoming flatter and bigger during passaging.Only 20%cells of passage 2 had mild swelling endoplasmic reticulum and demyelinate mitochondrion.More swelling endoplasmic reticulum and demyelinate mitochondrion were observed by scanning electron microscope from passage 3.The proliferation of the cells slowed from the 6^thpassage and almost stopped at the 8^thor 9^thpassage.The positive rate of senescence associated β-galactosidase staining and p16^INK4agene increased during passaging.Osteogenic and adipocytic potential were attenuated in cells after 6^thpassage.Conclusions: MSCs enter senescence during long term culture.Their potential of proliferation and multipotency dropped.The state of senescence should be considered for culture expanded MSCs when they are used for study.