Human CD34+ Cord Blood Cells Ex Vivo Expanded By TAT-HOXB4 Protein Can Repopulate NOD/SCID Mice Hematopoietic Ability

The human cord blood is an important source of hematopoietic stem cells both for transplantation and gene therapy application, which is about 0.5% CD34 positive cell in the cord blood. However, a single cord blood collection may not be sufficient to guarantee engraftment of adult allogeneic recipients.It is a great challenge to expanse hematopoietic stem cells, and the most useful way is the different cytokines or the gene transfection . It was interested in powerful intrinsic agonist of HSC self-renewal in vitro and in vivo. The human homeobox B4 (HOXB4) gene, a member of the homeobox gene(HOX) family, is an important and potent regulator in the early time of the hematopoiese, and the combined results argue in favor of the existence of certain threshold levels for HOXB4 activity that control the differentiation and self-renewal behavior of hematopoietic stem and progenitor cells. HOXB4 gene code a kind of homeoprotein binding to special DNA sequences, of which can get into the cell membrane by endosmosis.Retroviral expression of HOXB4 in mice significantly improved HSC regeneration in vivo, with three log increases of HSCs in both primary and secondary recipients without affecting normal differentiation or inducing cell transformation.Recombinant hoxb4 (TAT-HOXB4)has been produced in order to avoid the potential toxicities of retroviral vectors. It is very essential to check the capability of the TAT-HOXB4 protein getting in to the intracellular. The first step is detecting the capability of the TAT-HOXB4 protein into the intracellular with GFP or western blotting. First marking the TAT-HOXB4 protein with GFP, at the same time dying the cell-nuclear with DAPI, then we could get the picture that the TAT-HOXB4 protein marking with the GFP was coincident with the picture of cell-nuclear dying with the DAPI. The western blotting shows that the nuclear contained the TAT-HOXB4 protein when the cells cultured with it, on the other hand the nuclear without TAT-HOXB4 protein. So we decided the TAT-HOXB4 protein was able to get into the cell membrane by endosmosis freely.Human cord blood CD34+ cells were cultured at 105 cells for 4 d with or without 15 nmol/l of the recombinant TAT-hoxb4-H protein. Cells were harvested and injected into sub-lethally irradiated (2.5 Gy) non-obese diabetic severe combined immunodeficient (NOD-SCID) mice.Recipients were euthanized 12 weeks after transplantation and BM nucleated cells were analysed for the presence of human CD45+ cells by flow cytometry. Mice were considered positive for human HSC engraftment when at least 0.1% CD45+ human cells were detected in the mouse BM cells. cells from positive mice were further analysed by flow cytometry using antibodies for human CD14, CD15, CD19 and CD34 antigens.Conclusion :The TAT-HOXB4 can effectively expand UCB CD34+ cells ,and the expanded cells can engraft the BM of NOD/SCID mice and reconstitute hematopoiesis.