Thrombopoietin does not induce lineage-restricted commitment of Mpl-R expressing pluripotent progenitors but permits their complete erythroid and megakaryocytic differentiation.
In this study, we examined the in vitro and in vivo effects of forced expression of Mpl-R (the thrombopoietin receptor) on the progeny of murine hematopoietic stem cells. Bone marrow cells from 5-FU-treated mice were transduced with retroviral vectors containing the human Mpl-R cDNA, or the neomycine gene as a control. After 7 days cocultivation on virus-producer cells, GpE86-Mpl-R or Gp86-Neo, the types of hematopoietic progenitor cells responding to thrombopoietin (TPO) were studied by clonogenic assays. Mpl-R-infected cells gave rise to CFU-GEMM, BFU-E, CFU-MK, but not CFU-GM while Neo-infected cells produced only megakaryocytic colonies. In addition, when nonadherent cells from GpE86-Mpl-R cocultures were grown with TPO as the only stimulus for 7 days, a marked expansion of CFU-GEMM, BFU-E, and CFU-MK was observed, while no change in CFU-GM number was seen. Erythroid and megakaryocytic maturation occurred in the presence of TPO while a block in granulocytic differentiation was observed at the myeloblast stage. The direct effects of TPO on Mpl-R-transduced progenitor cells were demonstrated by single cell cloning experiments. To analyze the effects of the constitutive expression of Mpl-R on the determination of multipotent progenitors (CFU-S) and long-term repopulating stem cells, Mpl-R- or Neo-infected cells were injected into lethally irradiated recipient mice. No difference was seen in (1) the number of committed progenitor cells contained in individual CFU-S12 whether colonies arose from noninfected or Mpl-R-infected CFU-S; (2) the mean numbers of progenitor cells per leg or spleen of mice reconstituted with Mpl-R- or Neo-infected cells, 1 or 7 months after the graft; and (3) the blood parameters of the two groups of animals, with the exception of a 50% reduction in circulating platelet counts after 7 months in mice repopulated with Mpl-R-infected bone marrow cells. These results indicate that retrovirus-mediated expression of Mpl-R in murine stem cells does not modify their ability to reconstitute all myeloid lineages of differentiation and does not result in a preferential commitment toward the megakaryocytic lineage.
Goncalves
F
F
Institut Gustave Roussy, INSERM, U362, Villejuif, France.
Lacout
C
C
Villeval
J L
JL
Wendling
F
F
Vainchenker
W
W
Dumenil
D
D
D000818:Animals; D001854:Bone Marrow Cells; D002454:Cell Differentiation / Q000187:drug effects; D019070:Cell Lineage; D002478:Cells, Cultured; D018920:Coculture Techniques; D003114:Colony-Forming Units Assay; D018076:DNA, Complementary / Q000235:genetics; D015672:Erythroid Precursor Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D005822:Genetic Vectors / Q000235:genetics; D006085:Graft Survival; D006410:Hematopoiesis / Q000187:drug effects*; D018380:Hematopoietic Stem Cell Transplantation; D006412:Hematopoietic Stem Cells / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D006801:Humans; D008297:Male; D008533:Megakaryocytes / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D051379:Mice; D008808:Mice, Inbred CBA; D009363:Neoplasm Proteins; D011518:Proto-Oncogene Proteins / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism; D011828:Radiation Chimera; D018121:Receptors, Cytokine; D053628:Receptors, Thrombopoietin; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012190:Retroviridae / Q000235:genetics; D013154:Spleen / Q000166:cytology; D013926:Thrombopoietin / Q000494:pharmacology*
D018076:DNA, Complementary; D009363:Neoplasm Proteins; D011518:Proto-Oncogene Proteins; D018121:Receptors, Cytokine; D053628:Receptors, Thrombopoietin; D011993:Recombinant Fusion Proteins; C082218:MPL protein, human; D013926:Thrombopoietin