Isolation Of Multipotent Cells From Human Term Placenta And Study On Its Biological Characteristic.

Objective Mesenchymal stem cells (MSCs) have the capability for high proliferation and self-renewal,which are widely distributed in a variety of tissues in the adult human body and produce a marked effect on the normal function and damage repair of human body's tissues and organs.They are the hot research field in regenerative medicine. Currently MSCs have been successfully isolated and identified from human bone marrow, peripheral blood, muscle, fat, cord blood, amniotic fluid and fetal tissues ,in which bone marrow mesenchymal stem cells(BMSCs) are studied mostly by the researchers who have made some progoress. However,it is difficult to collect bone marrow which would induce infection and complication,and the quantity, proliferation and differentiation capacity decreased significantly as the age increased.In recent years, some researchers have found that MSCs exist in the placenta. Compared with bone marrow,placenta as a temporary organ that is discarded after parturition,along with the ease of accessibility, lack of ethical concerns.Thus placenta may be an attractive source of mesenchymal stern / progenitor cells for basic and clinical application. This experiment tends to isolate a new population of multipotent cells from human term placenta(placenta-derived mutipotent cells,PDMCs),and to identify the biological characterization of these cells. Subject and Methods(1) Collecting 30 healthy pregnant woman mature placenta tissue under sterile condition.(2) Preparation of mononuclear cells(MNCs):Mononucleated cells were isolated from the humanplacenta tissue perfusate by density gradient fractionation.Individual colonies were selected and cultured in tissue dishes.(3)Use the transmission electron microscope(TEM) to detect the ultrastructure of PDMCs; the surface antigens markers of PDMCs are detected by FlowCytometry(FCM).Results(1) cells cuture: Cell colonies appear aooroximately 9-14days after initial plating. The fibroblastoid population of cells continued to proliferate,even after 15 passages.(2)The morphous of cells: PDMCs eshibited fibroblastoid and spind-shaped morphology by detecting with inverted phase contrast microscope and hematoxylin and eosin stain.(3) The cells growth pattern:The period from the 4^th day to the 7^th day is the log phase growth;The period from the 7^th day to 14^th day is stationary phase growth;After the 14^th day,the growth comes to the decline phase;Which resemble that of MSCs.(4)The ultrastructure of cells: Use TEM to detect ultrastructure both of PDMCs and BMSCs,and the result reveals that they have common featureas: nuclear membrane is irregular , nucleole is obvious; euchromatin is major, there are affluent rough endoplasmic reticulum (RER) ,mitochondria,and ribosome in kytoplasm ,as well as some microfilament. Cell membrane is integrity,and there are many microvilli on its surface.(5)Surface marker:Flow cytometric analyses and immunohistochemistry revealed that PDMCs were CD9,CD29,CD44, CD105,CD106,HLA-ABC positive,but were negative for CD34,CD45,and HLA-DR,which is common to BMSGs.Besides,PDMCs are postive for markers of ESCs such as SSEA-3,SSEA-4,TRA-1-60,TRA-10-81,Oct-4.Conclusion (1) There are MSCs common to BMSCs in term placenta.(2) PDMCs are more primitve than BMSCs.