title: Osteopetrotic mouse stroma with thrombopoietin, c-kit ligand, and flk-2 ligand supports long-term mobilized CD34+ hematopoiesis in vitro.
abstract: OP-9 cells are stromal cells derived from macrophage colony-stimulating factor (M-CSF)-deficient osteopetrotic mice. To evaluate the OP-9 capability to sustain long-term hematopoiesis, we reported the expansion of granulocyte colony-stimulating factor (G-CSF)-mobilized human peripheral blood (PB) CD34(+) cells in co-culture with murine OP-9 and MS-5 stromal cells, either transfected with various combinations of adenovectors (Ad) expressing c-kit ligand (KL) (either soluble or transmembrane form), thrombopoietin (TPO), flt-3/flk2 ligand (FL), and granulocyte-macrophage (GM)-CSF or with weekly addition of these cytokines. Expression of TPO as well as association of TPO, FL, and KL increased progenitor cell and week-6 cobblestone area forming cell (CAFC) production in all stromal co-cultures. Similar progenitor expansion was obtained by weekly addition of soluble cytokine. Five weeks of co-culture with OP9 and TPO, FL + KL resulted in the greatest expansion of progenitor cells and week-6 CAFC as measured by secondary assay on MS-5. In contrast to MS-5 and TPO or TPO + FL + KL cultures where hematopoiesis declined by week 4, progenitor as well as week-6 CAFC expansion continued for over 3 months in TPO + FL + KL OP9 cocultures. This was associated with decrease of CD14(+) macrophage production. The addition of human macrophage (M)-CSF or CD14(+) cells to the co-culture decrease progenitor and stem cell expansion; however, murine M-CSF to OP-9 co-cultures did not decrease progenitor expansion. High levels of stromal-derived factor-1 (SDF-1) production by MS-5 and low or absent production by OP-9 may account for stem cell adhesion and CAFC formation in the former cultures and the predominance of stem and progenitor cells in the nonadherent fraction in the latter cultures.
authors.affiliation: James Ewing Laboratory of Developmental Hematopoiesis, Memorial Sloan-Kettering Cancer Center, New York, NY 10021, USA. p.feugier@chu-nancy.fr
authors.forename: Pierre
authors.initials: P
authors.lastname: Feugier
authors.forename: Na
authors.initials: N
authors.lastname: Li
authors.forename: Deog-Yeon
authors.initials: DY
authors.lastname: Jo
authors.forename: Jae Hung
authors.initials: JH
authors.lastname: Shieh
authors.forename: Karen L
authors.initials: KL
authors.lastname: MacKenzie
authors.forename: J F
authors.initials: JF
authors.lastname: Lesesve
authors.forename: V
authors.initials: V
authors.lastname: Latger-Cannard
authors.forename: D
authors.initials: D
authors.lastname: Bensoussan
authors.forename: Ronald G
authors.initials: RG
authors.lastname: Crystal
authors.forename: Shahin
authors.initials: S
authors.lastname: Rafii
authors.forename: J F
authors.initials: JF
authors.lastname: Stoltz
authors.forename: Malcolm A S
authors.initials: MA
authors.lastname: Moore
mesh_terms: D000256:Adenoviridae / Q000235:genetics / Q000378:metabolism; D000276:Adjuvants, Immunologic / Q000378:metabolism; D000818:Animals; D018952:Antigens, CD34 / Q000235:genetics / Q000378:metabolism*; D018929:Cell Culture Techniques / Q000379:methods; D002460:Cell Line; D002633:Chemotaxis / Q000502:physiology; D018920:Coculture Techniques; D005822:Genetic Vectors; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000235:genetics / Q000378:metabolism; D006410:Hematopoiesis / Q000502:physiology*; D006801:Humans; D018950:Lipopolysaccharide Receptors / Q000235:genetics / Q000378:metabolism; D016173:Macrophage Colony-Stimulating Factor / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D010022:Osteopetrosis; D019089:Stem Cell Factor / Q000235:genetics / Q000378:metabolism*; D013234:Stem Cells / Q000166:cytology / Q000378:metabolism; D017154:Stromal Cells / Q000166:cytology / Q000378:metabolism*; D013926:Thrombopoietin / Q000235:genetics / Q000378:metabolism*
chemical_list: D000276:Adjuvants, Immunologic; D018952:Antigens, CD34; D018950:Lipopolysaccharide Receptors; D008565:Membrane Proteins; D019089:Stem Cell Factor; C084881:flt3 ligand protein; D016173:Macrophage Colony-Stimulating Factor; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D013926:Thrombopoietin