Transplantation of Human Peripheral Blood CD34-Positive Cells in Combination with Ex Vivo Expanded Megakaryocytes Results in Fast Platelet Formation in NOD/SCID Mice.
Authors: Tijssen Marloes R. , Van Hennik Paula B. , Di Summa Franca , Hordijk Peter L. , Zwaginga Jaap Jan , Van der Schoot C. Ellen , Voermans Carlijn 
Published: October 2019

Abstract
The period of severe thrombocytopenia after high dose chemotherapy may be shortened by the addition of ex vivo expanded megakaryocytes (MKs, CD41+ cells) to the graft. This has been shown for expanded cord blood stem cells in mouse models, but hardly any effect has been observed in clinical trials in PBSC transplantation. The aim of the present study is to show the fate, engraftment ability and platelet production capacity of ex vivo expanded megakaryocytes from human mobilized peripheral blood (MPB) in NOD/SCID mice.
MPB CD34+ cells were cultured for 7 days in the presence of Tpo (100 ng/ml) and IL-1 (10 ng/ml). Cell numbers increased approximately 6-fold after 7 days of culture. Over 50% of the expanded cells expressed CD41, and only a limited number of cells still expressed CD34. After sublethal irradiation (3.5 Gy), NOD/SCID mice were transplanted with unmanipulated CD34+ cells (group A), expanded MKs (Group E) or a combination (group B, C, D) [Table 1]. As a control, the mice of group F did not receive any cells after irradiation. Blood was collected at day 3, 7, 10, 14, 21, 28, and 35 after transplantation. After three days, human platelets could be detected in the blood of the mice from groups C and E. After 7 days, human platelets were detected in the blood of the mice from all groups that received a transplant, except for group A. In the mice of the groups A, B, C and D platelet numbers increased till day 14 (to an average of 6.9 × 10^6/ml blood) with a small decrease toward day 35 (1.3 × 10^6/ml). The mice of group E reached a maximum of 3.4 × 10^5 human platelets per ml blood at day 10 and numbers declined from thereon to be below threshold at day 21. At day 35, mice were sacrificed and the presence of human cells in the blood, lung, spleen and BM was determined. In the BM, the chimerism (CD45+ cells) correlated with the number of unmanipulated CD34+ cells transplanted per group. This was also observed for all other lineages tested in the BM and a similar pattern was found in the spleen and blood. However, the percentages of CD45+ cells in spleen and blood were lower than in BM. No human cells were detected in the lung. In summary, IL-1- and Tpo-expanded MKs can significantly contribute to thrombocytopoiesis during the first days after transplantation. This indicates that the period of thrombocytopenia after intensive chemotherapy can be overcome by the co-transplantation of ex vivo generated human MPB-derived MKs. It may therefore be interesting to extend our culture protocol to a clinical setting.
Group Cells Cells transplanted Mice (n) A MPB CD34-positive 4.5×106 NC/mouse 100% 4 B MPB CD34-positive + expanded CD34-positive 4.05×106 NC + 0.45×106 NC input culture/mouse 90% + 10% 4 C MPB CD34-positive + expanded CD34-positive 4.5×106 NC + 4.5×106 NC input culture/mouse 100% + 100% 4 D MPB CD34-positive + expanded CD34-positive 2.25×106 NC + 2.25×106 NC input culture/mouse 50% + 50% 4 E Expanded CD34-positive 4.5×106 NC input culture/mouse 100% 4 F Control - 3