Isolation And Identification Human Placenta Derived Mesenchymal Stem Cells And Its Effect On Umbilical Cord Blood Derived Hematopoietic Stem/Progenitor Cells Ex Vivo Expansion

Human bone marrow contains a rare population of mesenchymal stem cells (MSCs). Theycan be extensively expanded in vitro and when cultured under specific permissive conditions,retain their ability to differentiate into multiple lineages including bone, cartilage, tendon, muscle,nerve, and stromal cells. MSCs are of great therapeutic potential because of their ability toself-renew and differentiate into multiple tissues. Adult bone marrow derived MSCs engraft innumerous organs and differentiate along tissue-specific lineages when transplanted into fetalsheep. They migrate into areas of muscle degeneration to undergo myogenic differentiation inimmunodeficient mice. In humans, allogeneic bone marrow transplantation in children withosteogenesis imperfecta allowed engraftment of functional donor MSCs, resulting in creasedbone marrow density. As the precursor of bone marrow stromal cells, marrow derived MSCshave been shown expressed hematopoietic cytokines to support hematopoietic stem/progenitorcells (HSPC) expression ex vivo and reconstructed marrow microenviroment to improvehematopoietic engraftment rate and pace. Currently, bone marrow represents the main source of MSCs for both experimental andclinical studies. The use of bone marrow-derived cells is not always acceptable due to the highdegree of viral infection and the significant drop in cell number and proliferative/differentiationcapacity with age. Thus, the search for possible alternative MSCs sources remains to bevalidated. At present, cells with mesenchymal progeintor characterization were isolated from severalmesenchymal tissues such as muscle, bone, cartilage and tendon, these tissues were all originfrom mesoderm during embryogenesis. Placenta was composed of vessel, mesenchyma andtrophocyte, was derived from extraembryonic mesoderm, which developed into fetal吉林大学博士学位论文 中文摘要 appertaining including placenta, fetal member and umbilical cord. Since fetal appertaining contains a large deal of mesenchyma, we have proposed that pluripotent MSCs derived from extraembryonic mesoderm, but not recruited to differentiate, may reside in a quiescent state within the placenta throughout gestation. Our aim was to isolate and characterize MSCs in human placenta, which would possibly open a new and rich source of MSCs for experimental and clinical needs. Umbilical cord blood (UCB) represents a potentially attractive alternative source of HSPCfor patients who require allogenic stem cell transplatation. In adults, however, this approach hasbeen hampered by the small numbers of HSC available in a single UCB unit. In particular, thetime to neutrophil engraftment has been relatively long. Now using combination of early actingcytokines and adherent cell layer including MSC to expansion of cord blood stem cells is beingexplored. As the UCB HSC is different from BMSC, the feeder layers derived from marrow do not get the same approving effect on it. Besides taking charge of interchange between fetus and matrix, placenta also has a secretive function. Furthermore, placenta and UCB are homogeneous, the microenviroment of placenta is much better for UCB. Therefore we isolated the MSC from human placenta, identifies its biological characterization and expression of hematopoietic cytokines and used the MSC as a feeder layer to support the UCB derived CD34＋ cells expansion, in order to seek for another potential culture feeder layer in the UCB for CD34+ cells ex vivo expansion. First, by using perfusate method mononuclear cells were isolated from placenta of full-termpregnancies male infants. A density gradient was used in the isolation procedure to eliminateunwanted cell types that were present in the placenta perfusate. While plated at a low density(12,000/cm2), a small percentage isolated from the density interface of 1.073 g/ml, cells werescattered attached and grew as fibroblastic c...