Filamins Regulate Differentiation of Megakaryocytes and Platelets From Cultured Embryonic Stem (ES) Cells.

Abstract 1618

Filamins (Flns) A and B are major non-muscle actin binding proteins that play important roles in cross-linking cortical actin filaments into three-dimensional networks. In addition to their role as cytoskeletal scaffolding molecules, filamins are also known to bind more than 30 other proteins, regulating their subcellular location and coordinating their ability to signal. The role of filamins in hematopoietic stem cell differentiation, however, remains unclear, in part because gene-targeted mice lacking filamins die early on in embryonic development. To investigate the role of filamins A and B in the differentiation of embryonic stem cells (ESCs) along the megakaryocyte/platelet axis, we designed shRNA-containing vectors that targeted both FlnA and B under the control of either the CMV immediate-early promoter (CMV-FlnAB_Low), or an endogenous Rosa26 promoter (Rosa26-FlnAB_Low). Compared with wild-type ESCs, FlnAB_Low ESCs formed small, tightly packed undifferentiated colonies that expressed high levels of the ESC transcription factor, Nanog, and low levels of ERK activity – all indicators of an undifferentiated state. Embryoid prepared from FlnAB_Low ESCs, were allowed to differentiate, and examined for markers of mesoderm differentiation (Flk-1) and megakaryocyte differentiation (CD41). Whereas Day 6 EB-derived FlnAB_normal wild-type cells were 8% Flk-1 positive and 13% CD41 positive, Day 6 FlnAB_Low cells were 9% Flk-1 positive and only 4% CD41 positive, consistent with the notion that loss of Fln A and B results in a delay of mesoderm to hematopoietic differentiation. To evaluate the effect of Fln knockdown on the ability of the CD41-positive cells to further differentiate into megakaryocytes, form proplatelet extensions, and produce platelets, CD41 positive cells isolated from day 8 EBs were cultured in the presence of a thrombopoietin (TPO)-producing TERT stromal cell line. We found that FlnAB_Low CD41-positive cells formed far fewer and smaller megakaryocytes compared with their FlnAB_normal wild-type counterparts. Proplatelets derived from FlnAB_Low cells exhibited an abnormal, enlarged morphology with swellings and thick shafts that released platelets prematurely, yielding platelets that were nearly twice the size of those derived from FlnAB_normal cells. Taken together, we conclude that not only do filamins function prominently in hematopoietic cell differentiation, they also play an important role in platelet production, likely via their ability to by recruit and organize the necessary signaling molecules near the inner face of the plasma membrane.