title: Animal serum-free culture of purified human CD34+ cells: amplification of progenitors from G-CSF and GM-CSF-mobilized peripheral blood.
abstract: The isolation and culture of human CD34+ cells could have broad clinical application for hematologic support following high-dose chemotherapy or bone marrow transplantation. The need for reproducible, animal product-free conditions for the culture of progenitors is crucial to the widespread clinical implementation of ex vivo cell therapies. In these studies, we explored the use of animal serum-free (ASF) medium for the culture of isolated human bone marrow and peripheral blood CD34+ cells. In this ASF system, isolated CD34+ cells were cultured using a variety of different growth factor combinations. Such ASF culture conditions yielded equivalent to superior cell and progenitor growth when directly compared with culture containing 10% fetal calf serum (FCS). In cultures containing IL-1, IL-3, and stem cell factor, total cell numbers increased, on average, 33-fold over the first 2 weeks. On phenotypic analysis, the ASF cultures demonstrated sustained proliferation of CD33+ myeloid cells throughout the culture period. CD34+ cell numbers increased during the first 7-10 days of culture, with a mean 3.4-fold expansion. Concomitant with the CD34+ cell expansion was an average 8.2-fold expansion of colony-forming unit-granulocyte-macrophage (CFU-GM) and a 102.0-fold increase in burst-forming units-erythrocytes (BFU-E). Likewise, a mean 4929-fold expansion of CD41a+ megakaryocyte progenitors was observed in these CD34+ cultures. Different combinations of growth factors affected the fold increase in cell and progenitor number. When CD34+ cell cultures from normal healthy volunteers mobilized with either G-CSF or GM-CSF were compared, similar expansions of total cell and progenitor cells resulted. However, CD41+ cells expansions were greater in those samples from G-CSF-mobilized volunteers in every case tested. These studies established the feasibility of this ASF CD34+ cell culture system to generate a population of maturing progenitors for potential use in transfusion support during cytopenic periods following high-dose chemotherapy or bone marrow transplantation.
authors.affiliation: Applied Immune Sciences, Inc., Santa Clara, CA 95054, USA.
authors.forename: L R
authors.initials: LR
authors.lastname: Schain
authors.forename: S
authors.initials: S
authors.lastname: Jain
authors.forename: M
authors.initials: M
authors.lastname: Wysocki
authors.forename: M
authors.initials: M
authors.lastname: Hall
authors.forename: B
authors.initials: B
authors.lastname: Dadey
authors.forename: R
authors.initials: R
authors.lastname: Pennathur-Das
authors.forename: W
authors.initials: W
authors.lastname: Biddle
authors.forename: J
authors.initials: J
authors.lastname: Wolf
authors.forename: T B
authors.initials: TB
authors.lastname: Okarma
authors.forename: J S
authors.initials: JS
authors.lastname: Lebkowski
mesh_terms: D000818:Animals; D018952:Antigens, CD34 / Q000097:blood*; D019070:Cell Lineage; D002469:Cell Separation; D002478:Cells, Cultured; D016895:Culture Media, Serum-Free; D015672:Erythroid Precursor Cells / Q000187:drug effects; D016179:Granulocyte Colony-Stimulating Factor / Q000494:pharmacology*; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000494:pharmacology*; D019650:Hematopoietic Stem Cell Mobilization / Q000379:methods*; D006412:Hematopoietic Stem Cells / Q000187:drug effects* / Q000276:immunology; D006801:Humans; D016130:Immunophenotyping; D008533:Megakaryocytes / Q000187:drug effects; D012016:Reference Values; D015203:Reproducibility of Results
chemical_list: D018952:Antigens, CD34; D016895:Culture Media, Serum-Free; D016179:Granulocyte Colony-Stimulating Factor; D016178:Granulocyte-Macrophage Colony-Stimulating Factor