title: Bone marrow repopulation by human marrow stem cells after long-term expansion culture on a porcine endothelial cell line.
abstract: In vitro exposure of murine hematopoietic stem cells (HSCs) to cell cycle-inducing cytokines has been shown to result in a defect in the ability of these cells to engraft. We used a porcine microvascular endothelial cell (PMVEC) line in conjunction with exogenous interleukin (IL)-3, IL-6, granulocyte-macrophage colony-stimulating factor (GM-CSF), and stem cell factor (SCF) to expand human HSCs that express the CD34 and Thy-1 antigens but lack lineage-associated markers (CD34+Thy-1+Lin- cells). Ex vivo expansion of hematopoietic cells was evaluated in comparison to stromal cell-free, cytokine-supplemented cultures. Cells expressing the CD34+Thy-1+Lin- phenotype were detectable in both culture systems for up to 3 weeks. These cells were reisolated from the cultures and their ability to engraft human fetal bones implanted into SCID mice (SCID-hu bone) was tested. HSCs expanded in PMVEC coculture were consistently capable of competitive marrow repopulation with multilineage (CD19+ B lymphoid, CD33+ myeloid, and CD34+ cells) progeny present 8 weeks postengraftment. In contrast, grafts composed of cells expanded in stroma-free cultures did not lead to multilineage SCID-hu bone repopulation. Proliferation analysis revealed that by 1 week of culture more than 80% of the cells in the PMVEC cocultures expressing the primitive CD34+CD38- phenotype had undergone cell division. Fewer than 1% of the cells that proliferated in the absence of stromal cells remained CD34+CD38-. These data suggest that the proliferation of HSCs in the presence of IL-3, IL-6, GM-CSF, and SCF without stromal cell support may result in impairment of engraftment capacity, which may be overcome by coculture with PMVECs.
authors.affiliation: Hematology/Oncology Section, University of Illinois at Chicago, USA.
authors.forename: J E
authors.initials: JE
authors.lastname: Brandt
authors.forename: A H
authors.initials: AH
authors.lastname: Galy
authors.forename: K M
authors.initials: KM
authors.lastname: Luens
authors.forename: M
authors.initials: M
authors.lastname: Travis
authors.forename: J
authors.initials: J
authors.lastname: Young
authors.forename: J
authors.initials: J
authors.lastname: Tong
authors.forename: S
authors.initials: S
authors.lastname: Chen
authors.forename: T A
authors.initials: TA
authors.lastname: Davis
authors.forename: K P
authors.initials: KP
authors.lastname: Lee
authors.forename: B P
authors.initials: BP
authors.lastname: Chen
authors.forename: R
authors.initials: R
authors.lastname: Tushinski
authors.forename: R
authors.initials: R
authors.lastname: Hoffman
mesh_terms: D000818:Animals; D001853:Bone Marrow / Q000502:physiology*; D016026:Bone Marrow Transplantation; D002455:Cell Division / Q000502:physiology; D002460:Cell Line; D018920:Coculture Techniques; D004730:Endothelium, Vascular / Q000166:cytology*; D006412:Hematopoietic Stem Cells / Q000166:cytology*; D006801:Humans; D051379:Mice; D017154:Stromal Cells / Q000166:cytology; D013552:Swine; D013997:Time Factors