One of the goals of stem cell medicine is the production of platelets from stem cells for transfusion therapy. The process of platelet production from megakaryocytes is complex and depends on a wide spectrum of internal and external stimuli. The degree of cell polyploidization, amount of demarcation membrane system (DMS), and the cells capacity to form proplatelets are major determines of the quantity of platelets released by each megakaryocyte. Both polyploidization and proplatelet formation depend on the synchronous function of microtubules and actin/myosin. This complex machinery is regulated in part by the actions of the C-Myc and NF-E2 promoters. These promoters play a crucial role during polyploidization and proplatelet formation. We have examined the potential role of these promoters in the efficacy of various chemical and cell culture-based methods of driving hematopoietic stem cells to megakaryocyte differentiation and platelet production.
Human cord blood derived CD34+ cells were isolated and 5×104cells were cultured with thrombopoietin (TPO) and stem cells factor (SCF) for 12 days. Megakaryocytes were cultured along with reagents that inhibit distinct mechanisms of the cytokinesis process: Rho-Rock inhibitor, Y27632 (RRI); Src-inhibitor, SU6656 (SI); Nicotinamide (NIC); Aurora-B inhibitor, ZM447439 (ABI); and Myosin Light Chain Kinase Inhibitor (MLCKI). Combinations of reagents were used in order to determine their interactions and to maximize megakaryocyte ploidy. The DMS was analyzed and quantified with Di-8 ANEPPS in flow cytometry and morphology was studied with Electron Microscopy (EM). On day 12 proplatelets were analyzed with an inverted microscope and platelets were counted with an Advia 120 cell counter. Total RNA was extracted and analyzed for C-Myc and NF-E2 mRNA by QRT-PCR.
All treatments increased megakaryocyte ploidy, except MLCKI. RRI reached the highest ploidy (p=0.0007), followed by NIC (p=0.003), SI (p=0.026) and ABI (p=0.018). Combinations all significantly increased polyploidization; however the only combination that equaled RRI alone was the combination of all of the other inhibitors (p<0.0001). EM showed normal megakaryocyte structure. DMS quantification showed that higher ploidy megakaryocytes had more extensive DMS (p<0.02). Higher ploidy megakaryocytes released more proplatelets and platelets than control and low ploidy cells (p=0.01). Treatments that had the lowest increase proplatelet formation were ABI and the combination of NIC-SI-ABI-MLCKI. RRI-NIC resulted in the highest release of proplatelet formation. C-Myc gene expression was down-regulated in most of the treatments. NF-E2 expression was up-regulated in megakaryocytes treated with ABI and in the combination of NIC-SI-ABI-MLCKI, but not in other treatments.
RRI proved to be the most effective agent in driving megakaryocyte polyploidization. The summation of effects of all of the other cytokinesis inhibitors increased polyploidization only to the same extent as RRI. Light and EM imaging showed that the cultured megakaryocytes were morphologically normal. Higher ploidy megakaryocytes with an extended DMS were able to extend more proplatelets and release more platelets in culture, especially after treatment with RRI-NIC. Gene expression analysis showed that down-regulation of C-Myc in late stages of development was correlated with increased proplatelet formation and platelet release. NF-E2 up-regulation on day 12 was associated with ABI treatment and decreased proplatelet formation in cultured megakaryocytes.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.