title: Direct evidence for ex vivo expansion of human hematopoietic stem cells.
abstract: To characterize human hematopoietic stem cells (HSCs), xenotransplantation techniques such as the severe combined immunodeficiency (SCID) mouse repopulating cell (SRC) assay have proven the most reliable methods thus far. While SRC quantification by limiting dilution analysis (LDA) is the gold standard for measuring in vitro expansion of human HSCs, LDA is a statistical method and does not directly establish that a single HSC has self-renewed in vitro. This would require a direct clonal method and has not been done. By using lentiviral gene marking and direct intra-bone marrow injection of cultured CD34+ CB cells, we demonstrate here the first direct evidence for self-renewal of individual SRC clones in vitro. Of 74 clones analyzed, 20 clones (27%) divided and repopulated in more than 2 mice after serum-free and stroma-dependent culture. Some of the clones were secondary transplantable. This indicates symmetric self-renewal divisions in vitro. On the other hand, 54 clones (73%) present in only 1 mouse may result from asymmetric divisions in vitro. Our data demonstrate that current ex vivo expansion conditions result in reliable stem cell expansion and the clonal tracking we have employed is the only reliable method that can be used in the development of clinically appropriate expansion methods.
authors.affiliation: Research Center for Regenerative Medicine, Tokai University School of Medicine Bohseidai, Isehara, Kanagawa 259-1193, Japan. andok@keyaki.cc.u-tokai.ac.jp
authors.forename: Kiyoshi
authors.initials: K
authors.lastname: Ando
authors.forename: Takashi
authors.initials: T
authors.lastname: Yahata
authors.forename: Tadayuki
authors.initials: T
authors.lastname: Sato
authors.forename: Hiroko
authors.initials: H
authors.lastname: Miyatake
authors.forename: Hideyuki
authors.initials: H
authors.lastname: Matsuzawa
authors.forename: Masayuki
authors.initials: M
authors.lastname: Oki
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Miyoshi
authors.forename: Takashi
authors.initials: T
authors.lastname: Tsuji
authors.forename: Shunichi
authors.initials: S
authors.lastname: Kato
authors.forename: Tomomitsu
authors.initials: T
authors.lastname: Hotta
mesh_terms: D000818:Animals; D018952:Antigens, CD34 / Q000378:metabolism; D018929:Cell Culture Techniques / Q000379:methods; D049109:Cell Proliferation; D002478:Cells, Cultured; D018380:Hematopoietic Stem Cell Transplantation / Q000379:methods; D006412:Hematopoietic Stem Cells / Q000166:cytology / Q000378:metabolism*; D006801:Humans; D016086:Lentivirus; D051379:Mice; D016688:Mice, Inbred NOD; D016513:Mice, SCID; D014161:Transduction, Genetic; D014183:Transplantation, Heterologous
chemical_list: D018952:Antigens, CD34