title: Stromal fibroblast heparan sulfate is required for cytokine-mediated ex vivo maintenance of human long-term culture-initiating cells.
abstract: We have recently demonstrated that 50% of primitive human long-term culture-initiating cells (LTC-IC) are maintained for up to 8 weeks in stroma-dependent cultures in which progenitor-stroma contact is prevented (stroma noncontact), or when progenitors are cultured in medium conditioned by stromal feeders. This indicates that factors responsible for LTC-IC maintenance are present in soluble form in stromal supernatant (SN). Although the picogram concentrations of cytokines present in stromal SN can induce the differentiation of CD34+/HLA-DR- (DR-) cells to clonogenic cells (colony forming cells; CFC), they maintain only 10% of LTC-IC for 5 weeks, suggesting that factors other than these cytokines are required for LTC-IC maintenance. To characterize the factor(s) in stromal SN responsible for LTC-IC maintenance, we purified glycoproteins and proteoglycans (PG) from the SN of the LTC-IC supportive murine marrow stromal fibroblast cell line M2-10B4 by ion exchange high performance liquid chromatography (HPLC). Culture of DR- cells in a combination of M2-10B4-derived PG, but not glycoproteins and picogram concentrations of recombinant human interleukin-6 (IL-6), granulocyte colony-stimulating factor (G-CSF), stem cell factor (SCF), leukemia inhibitory factor (LIF), granulocyte-macrophage colony-stimulating factor (GM-CSF), and macrophage inflammatory protein-1alpha (MIP-1alpha) resulted in the recovery of 96% +/- 8% of LTC-IC maintained in cultures supplemented with unfractionated stromal SN. LTC-IC maintenance was largely retained after digestion of the PG-rich fraction with proteinase K and after dissociative gel filtration chromatography, but was completely abolished following treatment with nitrous acid, which digests heparan sulfate glycosaminoglycans (HS GAG). As for M2-10B4-derived HS GAG, high concentrations of bovine kidney HS GAG, but not bovine tracheal chondroitin sulfate, significantly improved cytokine-mediated LTC-IC maintenance. Maintenance of LTC-IC by these nonmarrow-derived HS GAG was, however, significantly lower than that seen with M2-10B4-derived HS. These studies demonstrate a role for marrow stroma-derived HS GAG in the long-term in vitro maintenance of human LTC-IC. Further structure-function analysis of these HS GAG may have important implications for ex vivo stem cell expansion and gene transfer into hematopoietic progenitors.
authors.affiliation: Department of Medicine, University of Minnesota, Minneapolis 55455, USA.
authors.forename: P
authors.initials: P
authors.lastname: Gupta
authors.forename: J B
authors.initials: JB
authors.lastname: McCarthy
authors.forename: C M
authors.initials: CM
authors.lastname: Verfaillie
mesh_terms: D000818:Animals; D001685:Biological Factors / Q000302:isolation & purification / Q000494:pharmacology; D001854:Bone Marrow Cells; D002417:Cattle; D018929:Cell Culture Techniques / Q000379:methods; D002454:Cell Differentiation / Q000187:drug effects; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D003238:Connective Tissue / Q000737:chemistry; D017077:Culture Media, Conditioned / Q000737:chemistry; D005347:Fibroblasts / Q000737:chemistry*; D016298:Hematopoietic Cell Growth Factors / Q000494:pharmacology*; D006412:Hematopoietic Stem Cells / Q000166:cytology* / Q000187:drug effects; D006497:Heparan Sulfate / Q000302:isolation & purification / Q000494:pharmacology*; D006801:Humans; D011509:Proteoglycans / Q000302:isolation & purification / Q000494:pharmacology; D011994:Recombinant Proteins / Q000494:pharmacology
chemical_list: D001685:Biological Factors; D017077:Culture Media, Conditioned; D016298:Hematopoietic Cell Growth Factors; D006497:Heparan Sulfate; D011509:Proteoglycans; D011994:Recombinant Proteins