title: Isolation of TPO-dependent subclones from the multipotent 32D cell line.
abstract: Using thrombopoietin (TPO), as selective pressure, several TPO-dependent clones were isolated from the murine multipotential IL-3-dependent cell line 32D. Four of them were fully characterized. They depended on TPO for survival and proliferation and, although retaining the capacity to grow in IL-3, did not respond to either EPO, G-CSF or GM-CSF. 32D TPO cells were heterogeneous in morphology and ranged from small cells, with a DNA content nearly tetraploid and a modal chromosome no. 66, to cells 50-75 microm in diameter containing multiple (up to 5-6) interconnected nuclei with a clear megakaryocyte (Mk) morphology by electron microscopy. Cell sorter isolation and single cell cloning experiments indicated that the small cells were those capable to proliferate in TPO and to generate the larger ones over time. 32D TPO cells expressed Mk-specific markers by FACS (CD41, CD61 and 2D5) and RT-PCR (acetyl cholinesterase E and platelet factor 4) and their unique profile, by gene array analysis, included expression of urokinase plasminogen activator surface receptor (CD87 or uPAR), plasminogen activator inhibitor and coagulation factor II (thrombin) receptor (Cf2r). In addition, by quantitative RT-PCR, 32D TPO clones expressed levels of Gata1 similar to those expressed by freshly isolated Mks (DeltaCt approximately 4.7 in both cases). In conclusion, the 32D TPO subclones described here are among the few pure Mk cell lines isolated so far and, for their unique properties, may prove themselves as a useful model to study Mk differentiation.
authors.affiliation: Department of Cell Biology and Neurosciences, Istituto Superiore Sanità, Rome.
authors.forename: Giovanni
authors.initials: G
authors.lastname: Amabile
authors.forename: Antonella
authors.initials: A
authors.lastname: Di Noia
authors.forename: Elena
authors.initials: E
authors.lastname: Alfani
authors.forename: Alessandro Maria
authors.initials: AM
authors.lastname: Vannucchi
authors.forename: Massimo
authors.initials: M
authors.lastname: Sanchez
authors.forename: Domenico
authors.initials: D
authors.lastname: Bosco
authors.forename: Anna Rita
authors.initials: AR
authors.lastname: Migliaccio
authors.forename: Giovanni
authors.initials: G
authors.lastname: Migliaccio
mesh_terms: D000818:Animals; D002460:Cell Line / Q000166:cytology* / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D002469:Cell Separation; D048429:Cell Size; D002470:Cell Survival / Q000187:drug effects; D002999:Clone Cells; D003115:Colony-Stimulating Factors / Q000494:pharmacology; D020869:Gene Expression Profiling; D016130:Immunophenotyping; D008533:Megakaryocytes / Q000166:cytology*; D051379:Mice; D039902:Multipotent Stem Cells / Q000166:cytology*; D013926:Thrombopoietin / Q000494:pharmacology*
chemical_list: D003115:Colony-Stimulating Factors; D013926:Thrombopoietin