Cultured CD41a+ Megakaryocytes Derived from Cord Blood CD34+ Cells Are Activated Via STAT5 but Not STAT3.

Lengthy periods of thrombocytopenia and prolonged platelet recovery (typically, a side-affect of high-dose chemotherapy and/or radiation) have been associated with cord blood transplants. It maybe possible to alleviate the period of thrombocytopenia using cultured megakaryocytes derived from CD34+ stem cells however, platelet development invitro is delayed. Using immuno-magnetic beads, CD34+ stem cells were isolated from Cord Blood (CB) units and then cultured in serum-free culture medium (X-VIVO-10®, BioWhittaker Co. Walkersville, MD) supplemented with Thrombopoietin Peptide Agonist (TpoA), Stem Cell Factor (SCF), Flt-3L, and Vascular Endothelial Growth Factor (VEGF). A series of culture experiments were setup to investigate the activation of the JAK-STAT pathway following cytokine stimulation. Using flow cytometry, CB CD34+ cell cultures were evaluated weekly for the expression of CD41a, STAT3, STAT5, and Proliferating Cell Nuclear Antigen (PCNA) at week 1–6 of culture. Typically, CD41a expression peaked at week 3 of culture. At week 3, the total cells were 14.1 ±4.9 x10⁶ cells per culture with 48.8 ±12.5% CD41a+ cells or 6.4 ±1.2 x10⁶ CD41a+ cells per culture. Similarily, PCNA activation peaked at week 3–4 (64.1 and 62.4%, respectively). The level of PCNA activation increased incrementally from weeks 1, 2 and 3 of culture however declined at 5 and 6 weeks of culture. However, there was an incremental increase in the activation of STAT5 during the 6 weeks of culture. The STAT5 expression did not peak until week 5–6 (80.9 and 73.8%, respectively). Conversely, STAT3 expression declined between week 1 and 6 from 3.2. to 0.4%, respectively. With the exception of weeks 1 and 2, there was only minor activation or no activation of STAT3. These results suggest that STAT5 but, not STAT3, is activated during CB megakarycyte differentiation. These results may provide incite as to CB megakaryocyte development and might explain the delayed production of platelets following transplant. The PCNA results suggest that the cultured cells reach their greatest proliferative potential at week 3 and 4 of culture. This might provide useful information as to the optimal time period for the transplant of the cultured cells and suggest that cells transplanted from 5 and 6-week cultures have less proliferative capacity post-transplant. Additional studies are needed to determine the differentiation of megakaryocytes and platelet development from CB CD34+ cells invitro.