Safety and Functional Studies of Ex Vivo Generated Megakaryocytic Progenitors/Platelets in a Myeloablative Non-Human Primate Model
Authors: Guan Xin , Qin Meng , Zhang Yu , Ren Zhihua , Jiang Wenhong , Ma Yupo , Dai Wei , Jiang Yongping 
Published: October 2019

Abstract
The ex vivo induction of megakaryocytes/platelets of hematopoietic stem cells represent an effective treatment for thrombocytopenia. We have previously reported a clinically feasible "two-stage culture system" for expanding CD34+ cells and producing megakaryocytes/platelets. With this culture system one cord-blood unit (2 × 106 - 5 × 106 CD34+ cells) yields sufficient megakaryocytes/platelets for treating up to 85 patients (with an average weight of 70 kg and infusion cell number 5.45 × 106 cells/kg). The induced human megakaryocytic cells were capable of producing functional platelets in xenotransplantation mouse model.
Here, we further tested the safety and feasibility of ex vivo generated megakaryocytic progenitors/platelets in a myeloablative non-human primate model. Mobilized peripheral blood CD34+ cells of cynomolgus monkeys (Macaca fascicularis) wereisolated after daily administration of G-CSF (100 μg/kg/day) and SCF (50 μg/kg/day) for five days. Expansion and subsequent differentiation of CD34+ cells and megakaryocytic progenitor cells were performed in a modified IMDM basal medium supplemented with various cytokine combinations including stem cell factor, Flt-3 ligand, thrombopoietin, interleukin 3, StemRegenin 1, interleukin 6 for 8 days. The expansion and differentiation processes were closely monitored by flow cytometry for expression of cell surface markers. In vitro morphological identification and CFU assays were carried out for monkey megakaryocytic progenitors. Furthermore, safety and efficacy of induced megakaryocytic progenitor cells were evaluated in vivo by using a thrombocytopenia model of monkeys. Briefly, cynomolgus monkeys were administered with carboplatin at a dose of 8 mg/kg/day on days 1, 2 and 3. On day 7 after the first carboplatin injection, the experimental group monkeys were infused with megakaryocytic progenitor cells (4.6±1.2 × 106/kg ) labeled with anti-monkey IgG-microbead-FITC conjugates. Negative control group was treated with normal saline. Peripheral blood and bone marrow from the tested animals were obtained for analyzing cell differentiation at various times. In vivo bleeding time was recorded to assess the matured platelet function, which was the time length of the bleeding to stop by making a cut in the forearm of the monkeys.
After culturing for 8 days, total cells were expanded up to 13.65±3.79-fold. Analysis of the percentage of component cells further showed that CD34+, CD41+, and CD34+/CD41+ megakaryocytic progenitor cells were 46.8%±3.2%, 22.9%±4.6%, and 20.3%±2.8%, respectively. Induced megakaryocytic cells were morphologically distinguishable as they were much larger than CD34+ cells with apparent lobular nuclei. CFU analysis revealed that they increased 12±4.3-fold on day 8 as compared with day 1. In the experimental group, platelet count nadir occurred on day 14 or 15 with 40% of normal value and completely recovered to the normal value on day 26. On the other hand, the nadir of the platelet count in the control group occurred on day 19 or 20 with 20% of normal value and recovered to the normal on day 32. During the nadir phase (from days 14 to 20), the in vivo bleeding time of experimental group was considered normal (6-7.5 minutes) relative to untreated normal monkey values (range 5-7 minutes), whereas it was longer (~8.5 minutes) in control group. Likewise, the nadir of white blood cell (WBC) count (with 45% of the normal value) occurred on day 17, which completely recovered on day 22 for the monkeys infused with induced megakaryocytic progenitors/ platelets. On the other hand, the nadir of WBC count of the control group was 35% of normal value on day 19, which recovered to the normal on day 36. Fifteen days after infusion, flow cytometry and fluorescent microscope analyses showed that about 1% of fluorescent cells remained in bone marrow, indicating successful engraftment of CD34+ stem/progenitor cells in the infused cell preparation. The infused monkeys have survived with no apparent abnormalities for more than one year.
Combined, our results strongly suggest that functional human megakaryocytes/platelets can be produced in a large-scale from CD34+ cells for potential clinical application. More importantly, induced non-human primate megakaryocytic progenitors/platelets can be safely administrated to myelosuppressive monkeys, contributing to platelet recovery and early engraftment.

Disclosures
Qin: Biopharmagen. corp: Employment. Ren:Biopharmagen corp: Employment. Jiang:Biopharmagen.corp: Employment.