title: In vitro expansion of CD34+/CD41+ cells from human peripheral blood CD34+/CD41- cells: role of cytokines for in vitro proliferation and differentiation of megakaryocytic progenitors.
abstract: The aim of this study is to clarify the transitional change of the proliferation and differentiation of human peripheral blood CD34+ cells to megakaryocytic lineage, focusing on its clinical application. We developed a rapid system to purify human peripheral blood CD34+ cells from healthy volunteers, which produced CD34+ cells with a 90% purity. The purified CD34+ cells predominantly consisted of CD41- cells, and the rate of coexpression of CD41 was 0.6% +/- 0.5%. When the purified cells were cultured in liquid phase for 10 days in the presence of recombinant human stem cell factor (rSCF: a ligand for c-kit), interleukin-3 (rIL-3), and thrombopoietin (rTPO: a ligand for Mpl), the number of CD34+/CD41+ cells increased to 19% +/- 7% of total expanded cells on day 4 (4 days of liquid culture) and then gradually decreased to 2.2% +/- 0.6% on day 10. The absolute number of CD34+/CD41+ cells increased and reached a plateau on day 6, and 1.7 +/- 0.6 x 10(5) CD34+/CD41+ cells were produced by 1 x 10(5) CD34+/CD41- day 0 cells. The CD34-/CD41+ cells appeared on day 6, continuously increased in number until day 10, and constituted the main population of expanded cells on day 10, with a value of 38% +/- 18%. On day 10, 19.5 +/- 10.6 x 10(5) of CD34-/CD41+ cells were produced by 1 x 10(5) CD34+/CD41- day 0 cells. The deletion of rTPO from this cytokine combination decreased the number of CD34+/CD41+ and CD34-/CD41+ cells, after days 6 and 8, respectively. Day 0 cells required rIL-3 for promoting colonies containing megakaryocytes, whereas rTPO alone promoted almost no megakaryocytic colonies from day 0 cells. Thus, a combination of IL-3 and SCF expands CD34+/CD41+ cells from CD34+/CD41- cells, and TPO mainly acts to increase CD34-/CD41+ cells. This study suggests that if the expansion of CD34+/CD41+ is performed in vitro, the 6 days' culture of peripheral blood CD34+/CD41- cells with a combination of IL-3 and SCF with TPO provides the most rapid and stable products of CD34+/CD41+ cells for the rapid recovery of platelets in patients with peripheral blood stem cell transplantation.
authors.affiliation: Department of Internal Medicine II, Hokkaido University School of Medicine, Sapporo, Japan.
authors.forename: K
authors.initials: K
authors.lastname: Koizumi
authors.forename: K
authors.initials: K
authors.lastname: Sawada
authors.forename: M
authors.initials: M
authors.lastname: Yamaguchi
authors.forename: A
authors.initials: A
authors.lastname: Notoya
authors.forename: T
authors.initials: T
authors.lastname: Tarumi
authors.forename: H
authors.initials: H
authors.lastname: Takano
authors.forename: Y
authors.initials: Y
authors.lastname: Fukada
authors.forename: M
authors.initials: M
authors.lastname: Nishio
authors.forename: E
authors.initials: E
authors.lastname: Katagiri
authors.forename: T
authors.initials: T
authors.lastname: Yasukouchi
authors.forename: N
authors.initials: N
authors.lastname: Sato
authors.forename: S
authors.initials: S
authors.lastname: Sekiguchi
authors.forename: T
authors.initials: T
authors.lastname: Koike
mesh_terms: D000328:Adult; D018952:Antigens, CD34 / Q000032:analysis* / Q000096:biosynthesis; D002454:Cell Differentiation / Q000187:drug effects; D002455:Cell Division / Q000187:drug effects; D003115:Colony-Stimulating Factors / Q000502:physiology; D016207:Cytokines / Q000502:physiology; D006412:Hematopoietic Stem Cells / Q000166:cytology; D006801:Humans; D007377:Interleukin-3 / Q000494:pharmacology; D007963:Leukocytes, Mononuclear / Q000166:cytology* / Q000276:immunology*; D008533:Megakaryocytes / Q000166:cytology; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000032:analysis* / Q000096:biosynthesis; D011994:Recombinant Proteins / Q000494:pharmacology; D019089:Stem Cell Factor / Q000494:pharmacology; D013926:Thrombopoietin / Q000494:pharmacology; D013997:Time Factors
chemical_list: D018952:Antigens, CD34; D003115:Colony-Stimulating Factors; D016207:Cytokines; D007377:Interleukin-3; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D011994:Recombinant Proteins; D019089:Stem Cell Factor; D013926:Thrombopoietin