Construction of humanized blood mouse models using human cord blood CD34⁺ cells by yolk sac transplantation

Background: Compared with utero transplantation in large animals such as goat, the utero heterogeneous hematopoietic stem cell transplantation in small animals such as mouse is facing difficulties. Rebuilding rate of heterogeneous hematopoietic stem cells in peripheral blood was below 1%. Objective: To observe rebuilding rate of heterogeneous hemopoiesis following human cord blood hematopoictic stem cell transplantation in mouse yolk sac, and to compare with outcomes of abdominal transplantation. Design, time and setting: The cytology in vivo experiment was performed at the Laboratory of Stem Cells and Regenerative Biology, Beijing University in October 2007. Materials: Cord blood from healthy newborns was collected at the Maternal and Child Health Hospital of Beijing Haidian District. ICR mice were obtained from Animal Experimental Center of Medical Department of Peking University, including 8 mice at gestational day 8.5, and 9 mice at gestational day 12.5. Phycocrythrin labeled anti-human CD45 monoclonal antibody and flow cytometer were purchased from BD. Methods: Human cord blood mononuclear cells were harvested by Ficoll method. CD34⁺ cells were purified and collected using magnetic bead sorting. Mice at gestational day 8.5 were anesthestized prior to abdominal cavity was opened to expose uterus. Under a microscope, 5-10 μ L cord blood CD34⁺ cells (5×10⁴) were harvested, and slowly injected into the yolk sac of fetus. Under the same operation condition, the same volume of cord blood CD34⁺ cells were injected into the abdominal cavity of mice at gestational day 12.5. Needles were rapidly taken. Pregnant mouse uterus was homing, and then the abdominal cavity was sutured for continued pregnancy, waiting for parturition. At 3 months after birthing, blood was obtained via the caudal vein. In the dark, phycoerythrin labeled anti-human CD45 monoclonal fluorescent antibody was added. Flow cytometry was used to measure CD45 expression. Rebuilding effect of human blood cells in mice in vivo was detected by positive rate. Main outcome measures: Effects of different transplanted methods on hematopoiesis reconstitution. Results: Totally 52 healthy mice were born by yolk sac injection of CD34⁺ cells. Three month later, 86.5% (45/52) mice presented CD45 positive reaction, with a high chimerism rate of 4.34%. Totally 48 healthy mice were born by abdominal cavity injection of CD34⁺ cells. Three month later, 81.3% (39/48) mice presented CD45-positive reaction. The chimerism rate was significantly lower compared with the yolk sac injection (t=2.363, P < 0.05). Conclusion: Yolk sac transplantation in an early stage can obtain a long-term reconstruction of hematopoietic stem cells in mice, and the outcome is better than the abdominal cavity transplantation. It also confirmed the possibility of establishing humanized blood mouse models with human cord blood hematopoietic stem cells using yolk sac transplantation.