Initial Construction Of The Human-to-swine Hematopoietic Chimera Model And The Research Of Molecular Incompatibility

Insufficient blood source and blood contamination are the major problems which have been puzzling clinical transfusion therapy for a long time. The research on blood substitutes, especially the erythrocyte substitutes, has not been made great progresses due to the limitation of current medical technology. The construction of human/animal hematopoietic chimera may provide a new feasible way to solve the problems. The human/animal hematopoietic chimera could be made by transplanting human hematopoietic stem cells (hHSC) into the radiated or new-born animal, in which HSC can settle down, proliferate and differentiate. The hematopoietic functions in chimera should be performed by human HSC and the blood cells should be partly or wholly originated from human HSC. Therefore, the blood cells in chimera should be similarly or completely in gene origin, cell phenotype and biological function to these in human body, and be theoretically the ideal resource to substitute human blood.Swine is recognized as one of the best xeno-special organ resources. The immuno- deficiency or suppression of the new-born swine can be used to avoid the restriction of immune barricade which inhibit or reject the formation and stabilization of HSC in human-to-swine hematopoietic chimera (human-swine HC). However, the genus differences of some bioactive molecules are presented to lead abnormal corresponding physiologic functions in xeno-animal body during the process of xenotransplantation. This phenomenon is called as the molecular incompatibility which interfere the formation and stabilization of the human-swine HC. In this research, we tried to construct human-swine HC by transplanting human cord blood HSC into neonatal swine and study the molecular incompatibility during the process of human HSC differentiating in swine hematopoietic microenvironment model in vitro and chimera.The contents and results of the research were as follows:First, we primarily cultured the tissues of bone marrow, adrenal gland, kidney, spleen, liver and skeletal muscle of the miniature swine and collected the medium respectively. The levels of of the key hematopoietic factors (SCF, IL-3, IL-6, GM-CSF, EPO) in medium were measured by quantitative ELISA. The results indicated the concentrations of SCF,IL-3,IL-6,GM-CSF,EPO in the medium of kidney were higher than those of several tissues(P<0.01) and were on the top at the time point of 24h except for GM-CSF which concentration reached to the top at last. Therefore, the conditioned medium of kidney at the time point of 24h was selected to prepare swine key hematopoietic factors compounds by low-temperature, high-osmotic pressure condensing techniques.Second, the miniature swine bone marrow was collected and the mononuclear cells were isolated by density gradient centrifugation. These mononuclear cells were inoculated in specific medium and formed adherence fibroblast-like cell layer, which was radiated by appropriate ray to imitate swine hematopoietic microenvironment in vitro. The CD34+ cells were sorted from human cord blood by magnetic activated cell sorting (MACS) technique. Then, the erythroid semi-solid culture system and the erythroid liquid culture system were respectively compared as the detection systems to analyse the molecular incompatibility of the human/swine key hematopoietic factors. The human erythroid progenitor cells were added to swine hematopoietic microenvironment model in vitro to induce its differentiation into the red blood cells. The results showed the promoting differentiation effect by swine hematopoietic microenvironment model was similar to this by human hematopoietic microenvironment model in vitro and suggested swine hematopoietic microenvironment model could provide the support role to human stem/progenitor cells differentiating into erythroid cells.Third, the human cord blood CD34+ cells (5×105/ Kg) were transplanted into neonatal swine through intraperitoneal injection, human SCF (50ng/ Kg.3d) and EPO (100U/Kg.3d) were also injected simultaneously. Peripheral blood (PB) was collected on day of 10, 20, 30, 40, 50, and 60 after injection. FACS was used to quantitate human cells in the chimera PB. The same-aged miniature swine PB was regarded as the negative control. These results indicated the experimental swines grew normally and had no infection and death. The FACS results showed the rate of human CD71+ cells in chimera swine PB reached the highest on the day of 20 after injection, and then decreased gradually. The genus-antibody against human cells could be detected on the age of 16d in the normal miniature swine PB, and on the age of 50d in the chimera swine PB.The cord blood hematopoietic stem cells were free from immunogenicity and were regarded as the wide spread-application-perspective stem cells. Owing to the immunodeficiency, neonatal swine has immunotolerance to herterologous antigen. Moreover, swine was similar to human in gene spectrum and can imitate human physiological process. Accordingly, neonatal swine is ideal experimental animal. Our experiments in vitro showed swine hematopoietic microenvironment could provide the support role to human stem/progenitor cells differentiating into erythroid cells under the simulation of human EPO. Experiments in vivo indicated human hematopoietic stem/progenitor cells could grow and differentiate into erythroid cells in chimera. Therefore, neonatal swine could be used to construct human-to-swine hematopoietic chimera and study the bio-characteristic of human HSC in chimera.