Investigation On The Capability Of Human Umbilical Cord Blood Stromal Cells To Support Hematopoiesis In Vitro

Introduction: HME is the situation of orientation, proliferation, differentiation and development of HSC, of which hematopoietic stromal cell is an important component. Hematopoietic stromal cell regulate the self-renewing, proliferation and differentiation by direct contact hematopotic cells, producing HGF and ECM to supporting the normal hematopoietic ability of body. The research and application on injury and repair of hematopoietic function in the past focus more on HSC, and it is needed to further study of the stromal cells.Hematopoietic stem cell in cord blood is more primitive to it in bone marrow and peripheral blood. It has several behaviors of large number of sources and convenient collection, light GVHD after engraftation, high proliferation and long-term bone marrow reconstruction. There is great promise for it's clinical application. Our prophase study have confirmed that there exist hematopoietic stromal cells and could effect amplification by definite cytokine combination in vitro. There have few investigation about whether or not the amplificated HUCBSC can repair hematopoietic injure and support hematopoietic reconstruction. In this study, we demonstrate that umbilical cord blood stromal cells have the capability to support hematopoietic reconstruction associated with their products-hematopoietic growth factors. In addition, direct contact can reconstruct hematopoiesis in vitro. we found that umbilical cord blood stromal cells can be resuscitated safely after stored in -80℃ in 5% DMSO,3%HES,4%HAS 180 days.Materials and methods:1.ELISA and RT-PCR test the expression of hematopoietic growth factors(TPO,GM-CSF and SCF) of human umbilical cord blood stromal cells. 2.Colculture CD34+ cell with human umbilical cord blood stromal cells, observing the capability of HUCBSC supporting colony formatting of CD34+ cell. 3. -80℃ cryopreserve HUCBSC, observing cryopreserved effect.Results:1.ELISA analyzing tests found that umbilical cord blood stromal cells excrete GM-CSF,SCF,TPO, get a peak in 7 days in vitro culture. The level of above three factors declines gradually after cultured 8 days in vitro.2. The results of RT-PCR show that umbilical cord blood stromal cells express GM-CSF,SCF and TPO. Furthermore, we found that umbilical cord blood stromal cells express higher level TPO than bone marrow stromal cells and express less GM-CSF,SCF.3.CD34+cells began proliferate after co-cultured with umbilical cord blood stromal cells for 4 days and formatted BFU-E, CFU-GM, CFU-Meg after co-cultured for 7 days. But BFU-E, CFU-GM,CFU- Meg decreased gradually after co-cultured for 14 days. Briefly, BFU-E, CFU-GM, CFU-Meg were much more than those of cultured without umbilical cord blood stromal cells.4.Human umbilical cord blood stromal cells sustain about 90% activity after cryopreserved in -80℃ in 5% DMSO,3%HES,4%HAS 180 days. They could support CD34+ cells format BFU-E, CFU-GM, CFU-Meg. Conclusion: Human umbilical cord blood stromal cells have the capability to support hematopoiesis and it is related to its secreted hematopoietic growth factors as well as direct contact. Moreover in vitro, we established a safety method cryopreservation umbilical cord blood stromal cells: -80℃ in 5% DMSO,3%HES,4%HAS.Our results indicate that umbilical cord blood stromal cells may possess potential utilized in hematopoietic reconstruction study.