Engraftment in NOD/SCID Mice of TPO Amplified CB Cells To Promote Platelet Development.

Abstract

A major disadvantage of human umbilical cord blood (CB) transplant is the delayed engraftment of platelets, which can lead to hemorrhagic complication, increased platelet transfusion requirements, prolonged alloimmunization and hospitalization. As a strategy for accelerating platelet recovery, the infusion of ex vivo expanded megakaryocytic progenitors to transplant patients has been proposed. NOD/SCID mice constitute an in vivo model to study human megakaryocytopiesis and platelet development after transplantation. CB was obtained from the placenta of full-term newborn, after informed consent provided according to the Declaration of Helsinki, CD34+ progenitors with amplified 100ng/ml thrombopoietin (TPO) were transplanted in NOD/SCID mice, and their ability to promote differentiation of the megakaryocytic lineage, with production of functional platelets was assessed. The engraftment was evaluated on bone marrow cells by flow cytometry analysis using human CD45 MoAb staining. Four weeks after transplantation, a mean value of 3.22% CD45+ human cells was obtained; this percentage significantly increased at week 8 (39.6% hCD45+ cells) and was observed to have remained constant at week 12 (45.6% hCD45+ cells). To evaluate the capacity of engrafted cells to produce platelets, we performed Facs analysis to monitor the appearance of human platelets in mouse peripheral blood. Mouse peripheral blood was collected via retro-orbital bleeding at different times (2, 4, 6 and 8 weeks) in EDTA-coated tubes. Platelet enriched plasma was analyzed after human CD41a-FITC staining by flow cytometry. Of the mice analyzed, 78.8% produced >0.1% human platelets. The results obtained with CB CD34+ cells amplified with TPO were compared with cells amplified in the presence of a cocktail of growth factors (100ng/ml KL, 50ng/ml FL, 10ng/ml IL6) containing TPO at low concentration (10ng/ml) (T10KF6) or high concentration (100ng/ml) (T100KF6). The mean engraftment values were 57.7% in the T10KF6 group and 10.0% in T100KF6 group. However, although reduced engraftment was obtained in T100KF6, a mean of 60.8% of mice produced (>0.1%) human platelets. In T10KF6 this percentage was reduced to 45.2%. Despite the strong variability in platelets production, a mean percentage of 0.49% human platelets was detected at week 2 after transplantation; at week 4, this percentage peaked, reaching 1.26% (which translated into 7.6×109/L total human platelet count). In some mice, a human platelet percentage of 7% (42.2×109/L) was obtained. The platelet count gradually decreased between weeks 6 and 8, with a mean of 1.02% (8.05×109/L) and 0.86% (7.99×109/L) respectively. Our results showed that TPO maintained about 2.2 times higher platelet count than T100KF6 and T10KF6 and that TPO might play an important role in the ex vivo expansion of haematopoietic cells, supporting the hypothesis that Mk lineage engraftment is capable of shortening time of human platelet recovery when transplanted in NOD/SCID mice.