ABSTRACT
BACKGROUND:Cord tissues,which immune system is relatively non-development with low expression of MHC,rely on the protection of the mother's immune system.Accordingly,studies underlying biological characteristics of umbilical cord mesenchymal stem cells (MSCs) arouse more and more attention.OBJECTIVE:To isolate,separate,and culture MSCs from the tissues of umbilical cord,and to explore the ability of multi-directional differentiation.DESIGN,TIME AND SETTING:The in vitro cytology experiment was performed at the Laboratory of Tissue Engineering of Sichuan University from October 2006 to May 2007.MATERIALS:The umbilical cord of healthy neonate with 37-40 weeks gestational age was obtained from delivery room of the Second West China Hospital,Sichuan University.METHODS:Pieces of umbilical cord were dissected along its long axis,vessel was pulled away,then cord was tied together with a suture creating "loops",then it was placed into collagenase solution,after 6-8 hours,suspended cells obtained from the loops suspension were centrifuged and cultured with adherent method,pass,aged when single cell-derived colonies were appeared.Finally,the 5th generation of cells was induced differentiation towards osteoblasts and lipoblasts.MAIN OUTCOME MEASURES:Morphology,growth,surface antigen expression,as well as the multiple differentiation potential of umbilical cord MSCs.RESULTS:The adherent cells obtained from umbilical cord after removal vessels showed short rod-like or fusiform shape,which can be proliferated and form single cell-derived colonies.Cells isolated from the umbilical cord high expressed integrin markers (CD29,CD51,CD71) but not hematopoietic lineage markers (CD34,CD45 and HLA-DR).These colonies can differentiate into osteoblasts that produce mineralized matrices,stained by alizarin red and alkaline phosphatase,and differentiate adipocytes that accumulate lipid vacuoles and are demonstrated by morphology,stained by oil red.CONCLUSION:Umbilical cord tissue contains a high number of MSC-like elements forming colonies that may be successfully expanded in culture.The proliferation colonies present matrix ceil immunophenotypes,and can differentiate into osteobiasts and adipocytes.
ABSTRACT
BACKGROUND: Compared with bone marrow-derived mesenchymal stem cells (MSCs),human umbilical cord blood (hUCB)-MSCs possess many advantages,including easy acquirement,low immunogenicity,able to tolerance a higher degree of HLA-matching inconsistency,and with higher purity.OBJECTIVE: To analyze the method and condition for in vitro isolation,purification,proliferation,and osteoblast and lipoblast differentiation of hUCB-MSCs.DESIGN,TIME AND SETTING: The present observational experiment was performed in the Key National Laboratory of Biological Treatment,Huaxi Hospital,Sichuan University (i.e.,Institute of Stem Cells and Tissue EngIneering) between September 2004 and November 2005.MATERIALS: Neonatal cord blood at gestational age 37 to 40 weeks.METHODS: hUCB-MSCs were collected in aseptic condition,isolated by density gradient centrifugation,sedimenting red cells with methylcellulose followed by density gradient centrifugation,or immunomagnetic beads negative technique of CD34+.Theisolated MSCs were used to carry on plastic adherent culture in L-DMEM +10% fetal bovine serum (FBS) or MesencultTM medium +10% FBS.The third passage of cells were used for surface antigen determination by flow cytometry and induced to differentiate towards osteoblasts and lipoblasts.MAIN OUTCOME MEASURES: ①Identification of hUCB-MSCs.②Confirmation of hUCB-MSC differentiation by alizarin red and oil red staining.RESULTS: The mononuclear cells isolated by sedimented and centrifuged way cultured in MesencultTM medium +10% FBS were most available.Obvious colonies appeared in the third passage.The cells obtained by only centrifugation in density gradient were hard to form colony,and those isolated by immunomagnetic beads were hard to culture.The surface antigens of these colony cells presented CD29,CD59,and CD71,hut did not express CD34,CD45,and HLA-DR,and so on.After alizarin red staining,osteoblast-differentiating colony cell cytoplasm exhibited mineralized matrices.After old red staining,lipoblast-differentiating colony cell cytoplasm was full of lipid vacuoles.CONCLUSION: The MSCs can be successfully isolated by sedimenting red cells with methylcellulose followed by centrifugation and cultured in MesencultTM medium +10% FBS.Obvious colony growth appears in the third passage.After induction,the MSCs can differentiate into osteoblasts and lipoblasts.