Study On The Culture And Amplification Of Human Stromal Cells Derived From Umbilical Cord Blood With Human AB Type Serum

Hematopoietic inductive microenvironment (HIM) is the site of implantation, proliferation, differentiation, and development for hematopoietic stem cells (HSC). As an important component of HIM, stromal cells have a close relationship not only with the self-renewal, proliferation, differentiation, and homing of HSC, but also with the occurrence, progress, and prognosis of hematological diseases. Human umbilical cord blood is an easily accessible source for stromal cells and human stromal cells derived from umbilical cord blood (hUCBDSCs) are generally believed to provide a high proliferative activity, low immunogenity in repair the function of injured HIM compared with the stromal cells from bone marrow. It has been reported that hUCBDSCs played an important role in the regulation of the self-renewing, proliferation and differentiation of hematopotic cells by direct contact, producing growth factors such as Thrombopoietin (TPO), Granulocyte Macrophage Colony Stimulating Factor (GM-CSF) and stem cell factor (SCF), inducing hematopotic cells'movement to the bone marrow,, producing extracellular matrix (ECM) to support HIM. However, the use of FBS during hUCBDSCs propagation carries the risk of transmission of know and unknown pathogens as well as xenoimmunization. And another obstacle for large scale production of hUCBDSCs is the small amount of stomal cells in the human umbilical blood sample. The ideal culture system would be under defined conditions devoid of animal sera, thus exclude the risks of its clinical application in patients. In our study, we have established, for the first time, a reliable culture system with human AB type serum for the propagation of hUCBDSCs,which provides a basis for its further application in clinic.MethodsHuman stromal cells were isolated from the umbilical cord blood by gradient isolation and immunomagnetic bead purification. The cells were cultured in the DMEM/F12 medium complemented with 15% human AB type serum, different concentration of bFGF (2.5-20μg/L) and 10-6mol/L cortisone. The effect of AB type serum in combination with different concentration of bFGF on the efficacy of colony-forming unit-fibroblast (CFU-F) was observed. The growth status and immunological phenotype of the stromal cells were studied.ResultsThe isolation, culture and amplification of hUCBDSCs were conducted successfully. The optimal culture condition for hUCBDSCs was recommended as DMEM/F12 medium supplemented with 15% human AB type serum, 10μg/L bFGF and 10-6mol/L cortisone. The hUCBDSCs exhibited positive immunostaining for Vimentin, CD34 and negative immunostaining for TdT,CD45,CK,which was in consistent with the reported character of human stromal cells.ConclusionThe hUCBDSCs could be cultured and propagated successfully with human AB type serum in vitro, which provide a foundation for further study on the clinical application of hUCBDSCs.