title: Distinct requirements for optimal growth and In vitro expansion of human CD34(+)CD38(-) bone marrow long-term culture-initiating cells (LTC-IC), extended LTC-IC, and murine in vivo long-term reconstituting stem cells.
abstract: Recently, primitive human bone marrow (BM) progenitors supporting hematopoiesis in extended (>60 days) long-term BM cultures were identified. Such extended long-term culture-initiating cells (ELTC-IC) are of the CD34(+)CD38(-) phenotype, are quiescent, and are difficult to recruit into proliferation, implicating ELTC-IC as the most primitive human progenitor cells detectable in vitro. However, it remains to be established whether ELTC-IC can proliferate and potentially expand in response to early acting cytokines. Here, CD34(+)CD38(-) BM ELTC-IC (12-week) were efficiently recruited into proliferation and expanded in vitro in response to early acting cytokines, but conditions for expansion of ELTC-IC activity were distinct from those of traditional (5-week) LTC-IC and murine long-term repopulating cells. Whereas c-kit ligand (KL), interleukin-3 (IL-3), and IL-6 promoted proliferation and maintenance or expansion of murine long-term reconstituting activity and human LTC-IC, they dramatically depleted ELTC-IC activity. In contrast, KL, flt3 ligand (FL), and megakaryocyte growth and development factor (MGDF) (and KL + FL + IL-3) expanded murine long-term reconstituting activity as well as human LTC-IC and ELTC-IC. Expansion of LTC-IC was most optimal after 7 days of culture, whereas optimal expansion of ELTC-IC activity required 12 days, most likely reflecting the delayed recruitment of quiescent CD34(+)CD38(-) progenitors. The need for high concentrations of KL, FL, and MGDF (250 ng/mL each) and serum-free conditions was more critical for expansion of ELTC-IC than of LTC-IC. The distinct requirements for expansion of ELTC-IC activity when compared with traditional LTC-IC suggest that the ELTC-IC could prove more reliable as a predictor for true human stem cell activity after in vitro stem cell manipulation.
authors.affiliation: Stem Cell Laboratory, Department of Molecular Medicine and Gene Therapy, Institute for Laboratory Medicine, University Hospital of Lund, Lund, Sweden.
authors.forename: V
authors.initials: V
authors.lastname: Ramsfjell
authors.forename: D
authors.initials: D
authors.lastname: Bryder
authors.forename: H
authors.initials: H
authors.lastname: Björgvinsdóttir
authors.forename: S
authors.initials: S
authors.lastname: Kornfält
authors.forename: L
authors.initials: L
authors.lastname: Nilsson
authors.forename: O J
authors.initials: OJ
authors.lastname: Borge
authors.forename: S E
authors.initials: SE
authors.lastname: Jacobsen
mesh_terms: D000818:Animals; D018952:Antigens, CD34; D018929:Cell Culture Techniques / Q000379:methods; D002454:Cell Differentiation; D002455:Cell Division; D016895:Culture Media, Serum-Free; D006410:Hematopoiesis; D019650:Hematopoietic Stem Cell Mobilization / Q000379:methods; D006412:Hematopoietic Stem Cells / Q000166:cytology* / Q000502:physiology; D006801:Humans; D051379:Mice
chemical_list: D018952:Antigens, CD34; D016895:Culture Media, Serum-Free