Extensive generation of human cord blood CD34+ stem cells from Lin−CD34− cells in a long-term in vitro system

Objective 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. Materials and Methods 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. Results In this culture system, more than 107 CD34+ cells can be continuously generated from 104 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. Conclusion 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.