Human Umbilical Cord Blood-derived Stromal Cells Promote Megakaryocyte Proliferation And Migration Through A Sdf-1-dependent Activation Of PECAM-1 Mechanism

Prolonged thrombocytopenia is a serious complication of high-dose chemotherapy followed by autologous or allogenic transplantation of hematopoietic stem and progenitor cells （HSCT）. Platelet transfusions are required to support the thrombopoietic recovery in treated patients. It has been shown that the need for platelet transfusion has drastically increased during the last few decades. However, platelet transfusions can augment the risk of transmission of infectious diseases and potentiate graft-versus-host disease. This support treatment of high-dose chemotherapy may also result in extra health care costs and patient inconvenience. So, the methods to restore the quantity and function of platelets efficiently and securely are remained to explore.Megakaryocytic development and thrombopoiesis are complicated biological process involving the proliferation and differentiation of megakaryocytic progenitor cells into immature megakaryocytes, as well as the development of immature megakaryocytes into mature megakaryocytes that in turn produce platelets. Another possible approach to abrogate or ameliorate post-transplant thrombocytopenia might be the transplantation of the induced megakaryocytes from megakaryocytic progenitor cells in vitro that would lead to the production of sufficient numbers of mature functional cells within days after transplantation.Hematopoietic inductive microenvironment （HIM）, the internal environment for sustaining and regulating the growth and development of hematopoietic cell, acts as an important role in the proliferation, differentiation and maturation of megakaryocytes. So, it is worth finding some methods from the reconstruction of HIM to cope with the injury of megakaryocytes.Bone marrow stromal cells （BMSCs） are the main component of hematopoietic inductive microenvironment. But it can’t be applied extensively in clinical practices for some reasons, such as the dysfunction of HIM in autotransplantation and histocompatibility in allograft. Besides, bone marrow extraction is a highly invasive procedure and the number and expansion capacity of BM stromal cells decline with increasing age. Based on some advantages, such as easy-to-obtain, convenient to collect and low immunogenicity, cord blood is now used as a new source of hematopoietic stem cells.Our group also has studied the human umbilical cord blood-derived stromal cells （hUCBDSCs） and the cord blood associated HIM for years. We have found the precusor cells of stromal cells exist in the cord blood. After the successful acquirement of hUCBDSCs in vitro in our preliminary experiments, bio-characteristics and hematopoietic supporting-capacity of these cells were observed. Moreover, as compared with the alternative expansion system, which BMSCs were used as the trophoblastic cells, the colony forming unit-megakaryocte rate was much higher in the co-culture system with hUCBDSCs to be trophoblastic cells in vitro; and the hUCBDSCs are more effective than the BMSCs to promote the CFU-Meg forming and recovery of platelets in radiated mice. It is hinted that hUCBDSCs might act as a special role for the proliferation of megakaryocytes.Although we have reported this biologic character of hUCBDSCs promoting the MK development effectively, its mechanism needs to be explained and explored. As we know, thrombopoietin （TPO） is main factor for MK development, but the application of TPO might result in bleeding caused by anticoagulated antibody formation. Stromal cell-derived factor-1 （SDF-1） is a chemotactic factor produced by stromal cells. It has reported that SDF-1 supported platelet production in TPO^-/- or mpl^-/-mice through interactions of MK progenitors with the BM vascular niche. It was found that MKs in PECAM-1^-/- mice cannot migrate along with the SDF-1 concentration gradient, and that these cells matured slowly. Based on the references reading and our preliminary experiments, we hypothesized that“the hUCBDSCs promote megakaryocytopoiesis through the influence of SDF-1 and PECAM-1”. To demonstrate that, we constructed megakaryocytes/hUCBDSCs co-culture model, observed the phenomenon of hUCBDSCs promoting MK development and focused on the SDF-1/PECAM-1 role in the mechanisms of hUCBDSCs to promote the proliferation and migration of megakaryocytes, especially the upstream and the downstream signal proteins or pathways of PECAM-1.Methods:This study was mainly focused on the following two aspects.1. To observe the influence of hUCBDSCs on the expression of PECAM-1 of megakaryocytic cell line HEL.This part of experiments includes: constructing the co-cultured model of hUCBDSCs and HEL cells; detecting the level of SDF-1 secreted by hUCBDSCs by ELISA; observing the influence of hUCBDSCs on the proliferation of HEL cells by CCK-8; observing the migrated effect of hUCBDSCs to HEL cells by transwell migration assay; detecting the expression of PECAM-1 of HEL cells in the co-cultured model by RT-PCR, Western blot and the Fluorescent immunostaining.2. To explore the SDF-1/PECAM-1 association roles in the MK development promoted by hUCBDSCsThis part of experiments includes two sections. The first section is to construct the RNAi lentivirus vectors of SDF-1 and PECAM-1, including:Designing the siRNA, setting up the vshRNA vectors, lentivirus packaging, transduction of target cells and detecting the efficiency of RNA interference.The second section is exploring the possible mechanisms of SDF-1/PECAM-1 association in HEL development, which includes:After knockdown of SDF-1 and PECAM-1, detecting the expression of PECAM-1 in HEL cells by RT-PCR and Western blot; detecting the expression of CXCR4 in HEL cells by RT-PCR, Western blot and Fluorescent immunostaining; observing the influence of hUCBDSCs on the proliferation of HEL cells by CCK-8 and cell cycle analysis; observing the migrated effect of hUCBDSCs to HEL cells by Transwell migration assay; detecting the protein level of SHP-2, phospho -Akt and phospho -ERK （phosphorylation of Akt and ERK） in HEL cells. Results:1. The hUCBDSCs secreted higher level of SDF-1 than hBMSCs, it got the peek value 3.5ng/ml at seventh day （p<0.05）. In the co-cultured model, hUCBDSCs promoted the proliferation of HEL cells, the OD numbers of CCK-8 test were higher than the control, both at 4th and 7th day（p<0.05）. From the outcome of migration analyses, the chemotaxis effect of hUCBDSCs to HEL cells was greater than the hBMSCs （p<0.05）. In this part , we emphatically found that hUCBDSCs can promote the expression of PECAM-1 in HEL cells, by the outcome of RT-PCR、Western blot and immunofluorescence staining.2. We constructed two RNAi lentivirous vectors of SDF-1 and PECAM-1. According to the outcome of RT-PCR and Western blot, they are proved to be effective to knock down the target genes.3.①Both at mRNA level and protein level, the expression of PECAM-1 in HEL cells increased after co-cultured with hUCBDSCs. But when the SDF-1 of hUCBDSCs was knocked down, the PECAM-1 level of HEL cells decreased. At the other hand, after the PECAM-1 of HEL cells was knocked down, the co-cultured model cannot restore the expression of PECAM-1 in HEL cells.②Knockdown of PECAM-1 didn`t affect the expression of CXCR4 in HEL cells, neither at the mRNA level nor the protein （p>0.05）.③In the co-cultured model, hUCBDSCs promoted the proliferation and migration of HEL cells （p<0.01）. But when the SDF-1 of hUCBDSCs was knocked down, the proliferation and migration of HEL cells were inhibited （p<0.01）. On the other hand, the PECAM-1 knockdown in HEL cells can result in the similar inhibited effect on the HEL cells proliferation and migration（p<0.01）.④In the co-cultured model, hUCBDSCs promoted the expression of SHP-2 in HEL cells. However the knockdown of SDF-1 and PECAM-1 respectively restrained the SHP-2 expression.⑤In the co-cultured model, hUCBDSCs promoted the phosphorylation of Akt and ERK in HEL cells. But the knockdown of SDF-1 and PECAM-1 respectively restrained the phosphorylation of Akt and ERK in HEL cells . Conclusions:1. HUCBDSCs secreted high level of SDF-1 and promoted the expression of PECAM-1 of HEL cells in co-cultured model.2. SDF-1/PECAM-1 association existed in HEL/hUCBDSCs co-cultured model and modulated the proliferation and migration of HEL cells.3. SDF-1/PECAM-1association can activate the pI3K/Akt and MAKP/ERK pathways to promote the development of HEL cells.