Infusion of a Cryopreservable Human Megakaryocyte-Biased Cell Product Results in Sustained Platelet Reconstitution In Vivo

The demand for platelet (PTL) transfusions has steadily increased, straining a supply that is limited by its dependency on donors, short lifespan, risk of infections and alloimmunization. This stimulated the search for alternative PTL sources including PTLs generated ex vivo from primary CD34⁺ cells and immortalized pluripotent stem cells. These approaches, however, are associated with obstacles such as scalability and encounter identical limitations as donor PTLs: short shelf life, storage at ambient temperature and limited lifespan after infusion. These obstacles lead us to focus our efforts on not producing PLTs but rather a cryopreservable cell product consisting of megakaryocytes (MK) that can produce PTLs after transfusion into patients.

Umbilical cord blood units (CBU) are readily available sources for stem cell for transplantation. We created an efficient and cost effective culture system in which CB-derived CD34⁺ cells are first expanded then allowed to mature into MKs. Initially, we determined the optimal culture period (10-11 days) resulting in the greatest number of CD41⁺/CD42b^- and CD41⁺/CD42b⁺ MKs which are capable of PTL production. Next, we used research and clinical grade CBU to generate clinically relevant doses of MK. The median number of CD34⁺ cells selected from one CBU was 2.5x10⁶ with a purity of 90% (n=4). Following expansion and MK maturation, these cells generated 5.8x10⁷ viable total nucleated cells (TNC)/CBU. Out of these, 3.3 x 10⁷ were CD41⁺ MKs which corresponds to a median cell dose of 4.1x10⁵ CD41⁺ cells/Kg of body weight. 92% of CD41⁺ MKs were mature CD42b⁺ cells which we previously showed that are capable of ex vivo platelet production. Finally, we performed clonogenic assays and found that one CBU can generate ~1.5x10⁶ CFU-MK.

One half of these MK-biased cultures was characterized and assessed immediately after culture and the other half was cryopreserved. The fresh product was infused into sublethally irradiated NSG mice and the presence of human PTLs in the mouse peripheral blood (mPB) was evaluated weekly for 8 weeks at which time the animals were also analyzed for hMK chimerism in the bone marrow (BM), spleen (SP) and lung. The results demonstrate that 87% of animals (13 out of 15) had a robust hPTLs population in their PB. hPTL were detected as early as week 1 post infusion and their number peaked on week 4 (median, 6x10³ hPTL/μl) after which it plateaued. The release of hPTL in the mPB was accompanied by the presence of hCD41⁺ MKs in the mBM, SP and the lung indicating that the infused cells provided both early hPTL release and a reservoir of MK precursors for continuous hPTL production. We also found that in addition to MKs, these same organs contained hCD34⁺, CD45⁺ and myeloid CD15⁺ cells. These findings underscore the capabilities of this product which might have broader clinical applicability such as utilization during myeloablative or suppressive chemo/radiotherapy to improve the time and duration for both neutrophil and platelet engraftment. Equally important, we provide novel evidence that the lung is a site for hMK engraftment after transplantation, in line with recent reports recognizing the pulmonary bed as site for platelet production in the mouse.

The major advantage of developing a MK-based product over ex vivo generated PTLs is the amenability of the former to cryopreservation thus becoming a readily available cellular therapy which would be amenable to stock-piling. Therefore, portions of the same MK products described above were cryopreserved then subjected to ex vivo and in vivo studies identical to these performed on their fresh counterparts. Following thawing, the average recovery rate was 71% for TNC and 74.3% for CD41⁺ cells. MK phenotype and morphology as well as the number CFU-MK generated ex vivo were identical to that found in the fresh product. Although the number of TNC in the thawed product was lower than that of its fresh counterpart, the number of hPTL detected after its infusion ranged from 0.4 to 20.5x10³ hPTL/μl which is comparable to that detected after infusion of the fresh equivalent, 0.7-16x10³ hPTL/μl.

In summary, we created a potent transfusable MK cell product that provides robust and sustained PTL and hematopoietic engraftment in vivo and maintains this capability after cryopreservation. Clinical development of such product is now being pursued for the treatment of thrombocytopenia in acute leukemia patients undergoing chemotherapy.