Extensive generation of human cord blood CD34(+) stem cells from Lin(-)CD34(-) cells in a long-term in vitro system.
Human CD34(-) hematopoietic stem cells (HSCs) have been identified as potential precursors of CD34(+) HSCs by using xenogeneic transplantation systems. However, the properties of CD34(+) cells generated from CD34(-) cells have not been precisely analyzed due to the lack of an in vitro system in which CD34(+) cells are continuously produced from CD34(-) cells. We conducted this study to determine whether CD34(+) cells generated in vitro from CD34(-) cells have long-term multilineage reconstitution abilities. Lin(-)CD34(-) population isolated from human cord blood was cultured in the presence of murine bone marrow stroma cell line, HESS-5, and human cytokines, thrombopoietin, Flk2/Flt3 ligand, stem cell factor, granulocyte colony-stimulating factor, interleukin 3 (IL-3), and IL-6. They were analyzed weekly for their surface markers expressions, colony-forming cells, long-term culture initiating cells (LTC-IC), and SCID repopulating cells (SRC) abilities up to 30 days of culture. In this culture system, more than 10(7) CD34(+) cells can be continuously generated from 10(4) CD34(-) cells over 30 days. These CD34(+) cells produce colony-forming units, LTC-IC, and SRC with multi-lineage differentiation, all of which are characteristic features of hematopoietic stem/progenitor cells. These findings suggest that CD34(-) HSCs have extensive potential for the generation of CD34(+) HSCs in vitro. This system provides a novel and potentially useful procedure to generate CD34(+) cells for clinical transplantation and gene therapy.
Ando
K
K
Department of Hematology, Tokai University School of Medicine, Kanagawa, Japan. andok@keyaki.cc.u-tokai.ac.jp
Nakamura
Y
Y
Chargui
J
J
Matsuzawa
H
H
Tsuji
T
T
Kato
S
S
Hotta
T
T
D000818:Animals; D018952:Antigens, CD34 / Q000032:analysis*; D000943:Antigens, Differentiation / Q000032:analysis; D001854:Bone Marrow Cells / Q000166:cytology / Q000502:physiology; D018929:Cell Culture Techniques / Q000379:methods*; D002454:Cell Differentiation; D019070:Cell Lineage; D002478:Cells, Cultured; D018920:Coculture Techniques; D003114:Colony-Forming Units Assay; D016207:Cytokines / Q000494:pharmacology; D005312:Fetal Blood / Q000166:cytology*; D006085:Graft Survival; D018380:Hematopoietic Stem Cell Transplantation; D006412:Hematopoietic Stem Cells / Q000737:chemistry / Q000166:cytology* / Q000187:drug effects; D006801:Humans; D051379:Mice; D016688:Mice, Inbred NOD; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017154:Stromal Cells / Q000166:cytology / Q000502:physiology
D018952:Antigens, CD34; D000943:Antigens, Differentiation; D016207:Cytokines