title: Cell-to-cell variability in the differentiation program of human megakaryocytes.
abstract: Differentiation of CD34(+) stem/progenitor cells into megakaryocytes is thought to be a uniform, unidirectional process, in which cells transform step by step from less differentiated precursor stages to more differentiated megakaryocytes. Here we propose the concept and present evidence based on single-cell analysis that differentiation occurs along multiple, partially asynchronous routes. In all CD34(+) cells cultured with thrombopoietin, surface appearance of glycoprotein IIIa (GPIIIa) preceded that of GPIb, indicating that the expression of these glycoproteins occurs in a timely ordered manner. Cellular F-actin content increased in parallel with GPIb expression. Only cells that expressed GPIb were polyploid, pointing to co-regulation of GPIb expression, actin cytoskeleton formation and polyploidization during megakaryocytopoiesis. On the other hand, most progenitor cells responded to thrombin but not to thromboxane A(2) analogue by rises in cytosolic [Ca(2+)](i). The appearance of thromboxane-induced responses during megakaryocytopoiesis was not strictly linked to glycoprotein expression, because cells showed responsiveness either before or after GPIb expression. The same non-strictly sequential pattern was observed for disappearance of the Ca(2+) response by prostacyclin mimetic; in some megakaryocytes it occurred before and in others after GPIb expression. Thus, megakaryocytic differentiation follows along independent routes that are either strictly sequential (GPIIIa and GPIb expression) or proceed at different velocities (Ca(2+) signal regulation).
authors.affiliation: Laboratory for Thrombosis and Haemostasis, Department of Haematology, University Medical Center Utrecht and Institute for Biomembranes, Utrecht University, The Netherlands.
authors.forename: Els
authors.initials: E
authors.lastname: den Dekker
authors.forename: Monique
authors.initials: M
authors.lastname: van Abel
authors.forename: Hans
authors.initials: H
authors.lastname: van der Vuurst
authors.forename: Guillaume J J M
authors.initials: GJ
authors.lastname: van Eys
authors.forename: Jan-Willem N
authors.initials: JW
authors.lastname: Akkerman
authors.forename: Johan W M
authors.initials: JW
authors.lastname: Heemskerk
mesh_terms: D018952:Antigens, CD34; D020013:Calcium Signaling; D002454:Cell Differentiation; D005312:Fetal Blood / Q000166:cytology; D006412:Hematopoietic Stem Cells / Q000166:cytology; D006801:Humans; D039661:Integrin beta3 / Q000032:analysis / Q000096:biosynthesis; D008533:Megakaryocytes / Q000166:cytology*; D008562:Membrane Glycoproteins / Q000032:analysis / Q000096:biosynthesis*; D019038:Platelet Glycoprotein GPIb-IX Complex / Q000032:analysis / Q000096:biosynthesis; D015203:Reproducibility of Results; D034061:Thrombopoiesis; D013926:Thrombopoietin / Q000494:pharmacology; D013997:Time Factors
chemical_list: D018952:Antigens, CD34; D039661:Integrin beta3; D008562:Membrane Glycoproteins; D019038:Platelet Glycoprotein GPIb-IX Complex; D013926:Thrombopoietin