Expansion in vitro of Transplantable Human Cord Blood Stem Cells Demonstrated Using a Quantitative Assay of their Lympho-Myeloid Repopulating Activity in Nonobese Diabetic-Scid/Scid Mice

• Published in: Proceedings of the National Academy of Sciences - PNAS 1997-09, Vol.94 (18), p.9836-9841
• Main Authors: Conneally, E., Cashman, J., Petzer, A., Eaves, C.
• Format: Article
• Language: English
• Subjects: Adult stem cells; Animals; Biological Sciences; Bone marrow; Cell Differentiation; Cell Division; Cell Movement; Cellular biology; Chlorofluorocarbons; Cultured cells; Diabetes; Fetal Blood - cytology; Hematopoietic Stem Cell Transplantation; Hematopoietic stem cells; Hematopoietic Stem Cells - cytology; Humans; Mice; Mice, Inbred NOD; Mice, SCID; Myeloid cells; Pluripotent stem cells; Progenitor cells; Rodents; Stem cells
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.94.18.9836

Human hematopoiesis originates in a population of stem cells with transplantable lympho-myeloid reconstituting potential, but a method for quantitating such cells has not been available. We now described a simple assay that meets this need. It is based on the ability of sublethally irradiated immunodeficient nonobese diabetic--scid/scid (NOD/SCID) mice to be engrafted by intravenously injected human hematopoietic cells and uses limiting dilution analysis to measure the frequency of human cells that produce both CD34-CD19+(B-lymphoid) and CD34+(myeloid) colony-forming cell progeny in the marrow of such recipients 6 to 8 weeks post-transplant. Human cord blood (CB) contains ≈ 5 of these competitive repopulating units (CRU) per ml that have a similar distribution between the CD38-and CD38+subsets of CD34+CB cells as long-term culture-initiating cells (LTC-IC) (4:1 vs. 2:1). Incubation of purified CD34+CD38-human CB cells in serum-free medium containing flt-3 ligand, Steel factor, interleukin 3, interleukin 6, and granulocyte colony-stimulating factor for 5-8 days resulted in a 100-fold expansion of colony-forming cells, a 4-fold expansion of LTC-IC, and a 2-fold (but significant, P < 0.02) increase in CRU. The culture-derived CRU, like the original CB CRU, generated pluripotent, erythroid, granulopoietic, megakaryopoietic, and pre-B cell progeny upon transplantation into NOD/SCID mice. These findings demonstrate an equivalent phenotypic heterogeneity amongst human CB cells detectable as CRU and LTC-IC. In addition, their similarly modest response to stimulation by a combination of cytokines that extensively amplify LTC-IC from normal adult marrow underscores the importance of ontogeny-dependent changes in human hematopoietic stem cell proliferation and self-renewal.