title: Early-acting cytokine-driven ex vivo expansion of mobilized peripheral blood CD34+ cells generates post-mitotic offspring with preserved engraftment ability in non-obese diabetic/severe combined immunodeficient mice.
abstract: The ability of ex-vivo expanded peripheral blood stem cells (PBSC) to engraft non-obese diabetic/severe combined immunodeficient (NOD/SCID) mice has not been evaluated to date. We investigated the maintenance of primitive SCID-repopulating cells (SRC) and long-term culture-initiating cells (LTCIC) in PBSC expanded with early-acting cytokines, thrombopoietin (TPO), stem cell factor (SCF) and FlT3-ligand (FL) with or without interleukin 3 (IL-3) and IL-6 in short-term (6 d) stroma-free serum-free cultures. TPO + SCF + FL and TPO + SCF + FL + IL-3 + IL-6 produced 5.9 +/- 1.97 and 18.25 +/- 4.49 (mean +/- SEM)-fold increase of CD34+ cells respectively. We tracked cellular division with PKH26 and sorted post-mitotic CD34+ PKH26(low) cells to assess their primitive functional properties. After culture with TPO + SCF + FL, LTCICs among post-mitotic cells increased 12.08 +/- 3.4 times, and 4.3 +/- 1.6 times when IL-3 + IL-6 were added. CD34+ PKH26(low) cells cultured with TPO + SCF + FL provided human multilineage (CD34, CD33 and CD19) engraftment in NOD/SCID mice, whereas no human cells were detected in mice injected with cells cultured with TPO + SCF + FL + IL-3 + IL-6. Percentages of CD34+/CD38-, CD34+/CD33-, CD34+/DR- and cells in G(0)/G(1) phase were similar among cells cultured with both cytokine combinations, indicating that the deleterious impact of IL-3 + IL-6 on the ability to engraft is not translated into phenotypic or cycling features. In conclusion, TPO + SCF + FL-expanded PBSC maintain multilineage engraftment ability in NOD/SCID mice, which is abrogated by the addition of IL-3 + IL-6.
authors.affiliation: Department of Haematology, Reina Sofia University Hospital, Avda. Menéndez Pidal s/n, 14004-Cordoba, Spain. md1togoa@uco.es
authors.forename: C
authors.initials: C
authors.lastname: Herrera
authors.forename: J
authors.initials: J
authors.lastname: Sánchez
authors.forename: A
authors.initials: A
authors.lastname: Torres
authors.forename: C
authors.initials: C
authors.lastname: Bellido
authors.forename: A
authors.initials: A
authors.lastname: Rueda
authors.forename: M A
authors.initials: MA
authors.lastname: Alvarez
mesh_terms: D000818:Animals; D018952:Antigens, CD34 / Q000276:immunology*; D016895:Culture Media, Serum-Free; D016207:Cytokines / Q000494:pharmacology*; D005434:Flow Cytometry; D006133:Growth Substances / Q000494:pharmacology*; D019650:Hematopoietic Stem Cell Mobilization / Q000379:methods*; D018380:Hematopoietic Stem Cell Transplantation; D007377:Interleukin-3 / Q000494:pharmacology; D015850:Interleukin-6 / Q000494:pharmacology; D008565:Membrane Proteins / Q000494:pharmacology; D051379:Mice; D016688:Mice, Inbred NOD; D016513:Mice, SCID; D008938:Mitosis; D019089:Stem Cell Factor / Q000494:pharmacology; D013234:Stem Cells / Q000276:immunology*; D013926:Thrombopoietin / Q000494:pharmacology
chemical_list: D018952:Antigens, CD34; D016895:Culture Media, Serum-Free; D016207:Cytokines; D006133:Growth Substances; D007377:Interleukin-3; D015850:Interleukin-6; D008565:Membrane Proteins; D019089:Stem Cell Factor; C084881:flt3 ligand protein; D013926:Thrombopoietin