Objectives

1. Effective megakaryocytic differentiation of umbilical cord blood hematopoietic stem/progenitor cells using two-phase protocolJavad Hatami, Pedro Z. Andrade, Joaquim M. S. Cabral, Claudia L. da Silva, Frederico Castelo Ferreira Department of Bioengineering and IBB - Institute for Biotechnology and Bioengineering – Instituto Superior Técnico (IST), Lisbon, Portugal Objectives Results show that over-expansion in 1st phase results in less efficient megakaryocytic differentiation with lower %CD41+ and EY. The process proves to be efficient with high EY, 48.17±7.71 Mk/CD34+, and 35±4.97 %Mk purity obtained for an intermediate FI-CD34+in the 1st stage. Results confirm the positive effect of using a feeder layer on the final Mk yield and purity (Fig.2).  Electron microscopy results confirm normal feature and morphology of culture-derived Mks and platelets (Fig.3).   There is a high need for the ex-vivo expansion of Hematopoietic stem and progenitor cells (HSC/HPC), especially those from the umbilical cord blood (UCB) which for their use in hematopoietic transplantation. Likewise, a high demand for cells with megakaryocytic potential exists aiming at the clinical scale production of platelets for transfusion. However the complexity of hematopoiesis, the heterogeneity of culture population and the different combination of culture parameters has impaired to establish an efficient protocol to be used for platelet production from UCB. Thus, we aimed to develop a two-phase ex-vivo protocol for expansion and differentiation of HSC/HPC from UCB towards megakaryocyte lineage using a static co-culture system. Methods After CD34+ enrichment by MACS, HSC/HPC from UCB were co-cultured with mesenchymal stem cells from bone marrow (BM-MSC) in a serum free medium (QBSF) with a cytokine cocktail including TPO, SCF, FLT3, b-FGF (50, 60, 55 and 5 ng/mL, respectively). After expansion, the hematopoietic cells were retrieved and submitted to a differentiation protocol using IMDM+10% FBS and a cocktail of TPO and IL3. Fig. 1) Effects of different concentration of TPO and IL3 on Mk differentiation. Results presented as mean ± SEM. Fig. 3) Electron microscopy Images: A. Representative scan electron microscopy images of plasma-derived platelet (left) and culture-derived platelet (right), B. TEM images of CD34+-derived Mk at 2nd phase in G2 group. FI-CD34+ = (total number of CD34+ cellst=day x)/(total number of CD34+cells t=0) Conclusions References EY = (total number of CD41+ cells) / (total number of CD34+ cells t= 0) • TPO (100)+IL3(10) is a simple cocktail that provides the best results for effective Megakaryocytic differentiation. • Expansion phase is critical for effective Megakaryocytic differentiation protocol, where the feeder layer and FI-CD34+ has the central roles. • Our simple protocol provides the expected morphology for in-vitro produced Megakaryocyte and Platelet. Results Results show increasing TPO concentration lead to the more pure population of Mks. However, synergic effect of TPO(100)+IL3 (10) provide higher EY but less %CD41+ compare to TPO (100) alone (Fig.1). Fig. 2) Effect of 1st expansion phase on the 2nd phase concerning (A) EY and (B) %CD41+.