Studies On Separation, Purification, Biologic Characters And B Cell Development Of Hematopoietic Stem/progenitor Cells From Cord Blood

ObjectiveThe aim of this study is to investigate the expansion of hematopoietic stem /progenitor cell separated by Ficoll in vitro and the potential of hematopoietic reconstitution in vivo,the role of expansion of CD34+ cells supported by fetal mesenchymal stem cells(MSCs) in vitro and the B cell development of hematopoietic stem/progenitor cells from cord blood in vitro.MethodsBy using a series of techniques,i.e.,stem/progenitor cells culture,expansion in vitro and animal transplantation model,the hematopoietic activity of different Ficoll-Urografin gradient density separated cells was evaluated.Fetal MSCs were isolated and purified in vitro.Cord blood CD34+ cells were isolated by using Mini-MACS separation system.Co-culture of CD34+ cells with fetal MSCs and cytokines was established.CD34+ cells was expanded in liquid culture.Nucleostemin gene in cultured cells was analyzed by Real-Time RT-PCR.Hematopoietic stem/progenitor cells from cord blood were isolated and purified by using immunomagnetic beads separation system in vitro.Differentiation of B cells derived from cord blood hematopoietic stem/progenitor cells supported by murine S-17 stromal cells were stimulated in co-culture with T3 and IL-7.The differentiation and development culture system of B cells were established in vitro.Differentiated B cells were detected by using FACS,PCR and RT-PCR techniques at different culture time.ResultsAfter separation of 1.064g/ml Ficoll-Urografin gradient,the number of CFU-GM were 373±289/1×10~5 MNCs and BFU-E were 121±70/1×10~5 MNCs.Following treated with cytokines for 14 days,the number of CFU-GM expanded 52.2-fold in vitro.The number of CFU-S in BALB/c mice which were irradiated by a lethal dose of 8.5Gy and transfused subsequently with the hematopoietic stem/progenitor cells separated by 1.064g/ml Ficoll-Urografin gradient was 2.2-fold as many as that of separated by 1.077g/ml Ficoll-Urografin gradient.Fetal MSCs expressed CD29 and CD44.The average purity of CD34+ cells was 97.4%.After co-culture of CD34+ cells with fetal MSCs for 28 days,6.43%of CD34+ cells remained in the cultured cells,the amplification of the total cells and CD34+ cells reached 1.65×10~5-fold,788-fold increase in the co-culture of CD34+ cells with fetal MSCs and cytokines,respectively.T3 stimulated differentiation of B cells derived from cord blood hematopoietic stem/progenitor cells,most of the induced CD19+ B cells were smaller,CD19+ B cells scarcely expressed CD21,development of B cells remained in a primitive stage. In experimental conditions we had selected,co-culture with T3,IL-7 and murine S-17 stromal cells was optimum condition to stimulated CD34+CD19- hematopoietic stem/progenitor cells to differentiate to B cells.Cultured CD19+ B cells expressed CD21 as well as CD10,CD20,CD24,weakly expressed CD23,HSL11,HSL96 and did not expressed CD3,CD33,CD34,IgM,B cells developed to be mature.Fold increase of B cells derived from CD34+CD19-CD38- hematopoietic stem/progenitor cells was significantly higher than that of CD34+CD19-hematopoietic stem/progenitor cells,the time of lasting amplification also extended.There was no significant difference in differentiation purity and amplification folds of B cells derived from fresh or cryopreserved cord blood.Murine S-17 stromal cells passaged more than 20 generations lost the ability of supporting the differentiation of B cells derived from cord blood CD34+CD19-stem/progenitor cells.The germline configuration of the DQ52 gene segment could be detected as indicated by its expected size of 2216bp.We found preferential rearrangements with a predicted fragment size of DH-JH4,DH-JH3,and DH-JH5 joined segments,whereas rearrangements to JH1,JH2 or JH6 gene segments were not detectable.Our primer design showed continuous transcription of Iμ,Cμ,IgLL during B cell development throughout the course of our in vitro cell culture system. ConclusionThe hematopoietic stem/progenitor cells after 1.064g/ml Ficoll-Urografin density separation acquired higher ability of expansion and sustaining hematopoiesis in vitro and the potential of hematopoietic reconstitution in vivo.Fetal MSCs could support efficiently proliferation of hematopoietic stem/progenitor cells derived from cord blood.In experimental conditions we had selected,supported by murine S-17 stromal cells,T3 and IL-7 could stimulate cord blood hematopoietic stem/progenitor cells to differentiate to B cells in vitro.There were preferential rearrangements of heavy chain immunoglobulin genes with DH-JH4,DH-JH3 and DH-JH5 joined segments.The activity of hematopoietic stem/progenitor cells and the ability of differentiation of B cells derived from hematopoietic stem/progenitor cells had no significant difference between fresh cord blood and cryopreserved cord blood.