title: Maintenance and self-renewal of long-term reconstituting hematopoietic stem cells supported by amniotic fluid.
abstract: The maintenance and self-renewal of hematopoietic stem cells (HSC) in culture is a central focus of hematopoietic stem cell research. In vivo, the balance between HSC differentiation, apoptosis, and self-renewal is regulated at the endosteal surface niche in the bone marrow (BM). In feeder-free cultures, the fate of HSC is affected by growth factors/interleukins and serum, which affect the balance between self-renewal, differentiation, and apoptosis and lead to the rapid loss of multipotent HSC. We report that substituting human amniotic fluid (AF) for serum in HSC cultures provides a growth milieu in which HSC differentiation and apoptosis are down-regulated and multipotent HSC are maintained. Murine BM cells were cultured in serum-free medium containing 25% amniotic fluid and stem cell factor (SCF) only, "AF/SCF" cultures. Compared with serum and multiple growth factor-containing medium, cells cultured for 4 weeks in AF/SCF medium displayed downregulation of differentiation markers while maintaining a high fraction of cells expressing Sca1 (51.8%) and c-kit (10.2%). Reconstitution of lethally irradiated C57BL/6 (Ly5.2) mice with cultured Ly5.1 BM cells resulted in high levels of (cultured) donor cells in primary (78 +/- 19.4% and 94.32 +/- 2.5%, 10(5) and 10(6) cells injected, respectively) and secondary (96.5%) recipients at 8 and 11 months post-transplantation. Hence, long-term repopulation with AF/SCF cultured BM cells was maintained. Addition to the cultures of 10% serum, interleukin (IL)-3, IL-6, granulocyte colony stimulating factor (G-CSF), or granulocyte-macrophage colony stimulating factor (GM-CSF), singly or in combination, resulted in rapid differentiation and apoptosis, leading to the total loss of HSC.
authors.affiliation: Department of Biology/Cancer Center, University of California, San Diego, La Jolla, CA 92093-0063, USA.
authors.forename: Emily
authors.initials: E
authors.lastname: Barria
authors.forename: Amanda
authors.initials: A
authors.lastname: Mikels
authors.forename: Martin
authors.initials: M
authors.lastname: Haas
mesh_terms: D000653:Amniotic Fluid / Q000166:cytology* / Q000378:metabolism; D000818:Animals; D017209:Apoptosis; D001854:Bone Marrow Cells / Q000166:cytology; D016026:Bone Marrow Transplantation; D018929:Cell Culture Techniques / Q000379:methods*; D002454:Cell Differentiation; D002469:Cell Separation; D002478:Cells, Cultured; D016895:Culture Media, Serum-Free / Q000494:pharmacology; D015536:Down-Regulation; D005260:Female; D005434:Flow Cytometry; D016179:Granulocyte Colony-Stimulating Factor / Q000378:metabolism; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000378:metabolism; D006412:Hematopoietic Stem Cells / Q000166:cytology*; D006801:Humans; D016130:Immunophenotyping; D007377:Interleukin-3 / Q000378:metabolism; D015850:Interleukin-6 / Q000378:metabolism; D008747:Methylcellulose / Q000737:chemistry; D051379:Mice; D008810:Mice, Inbred C57BL; D010641:Phenotype; D019009:Proto-Oncogene Proteins c-kit / Q000378:metabolism; D019089:Stem Cell Factor / Q000378:metabolism; D013234:Stem Cells / Q000166:cytology; D013997:Time Factors
chemical_list: D016895:Culture Media, Serum-Free; D007377:Interleukin-3; D015850:Interleukin-6; D019089:Stem Cell Factor; D016179:Granulocyte Colony-Stimulating Factor; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D008747:Methylcellulose; D019009:Proto-Oncogene Proteins c-kit