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

^*Terry Fox Laboratory, British Columbia Cancer Agency, and Departments of ^†Pathology and Laboratory Medicine and ^§Medical Genetics, University of British Columbia, Vancouver, BC Canada V5Z 1L3

Communicated by Ernest Beutler, The Scripps Research Institute, La Jolla, CA (received for review May 5, 1997)

Abstract

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 describe 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.

Footnotes

↵ ‡ E.C. and J.C. contributed equally to this work.
↵ ¶ To whom reprint requests should be addressed at: Terry Fox Laboratory, 601 West 10th Avenue, Vancouver, BC Canada V5Z 1L3. e-mail: connie{at}terryfox.ubc.ca.
ABBREVIATIONS:

NOD/SCID,

nonobese diabetic–scid/scid;

CRU,

competitive repopulating unit;

LTC-IC,

long-term culture-initiating cell;

CFC,

colony-forming cell;

CB,

cord blood;

SF,

Steel factor;

FL,

flt3-ligand;

IL,

interleukin;

CSF,

colony-stimulating factor;

CFU-GM,

colony-forming unit-granulocyte, macrophage;

CFU-GEMM,

colony-forming unit-granulocyte, erythroid, macrophage, megakaryocyte;

BFU-E,

burst-forming unit-erythroid;

FITC,

fluorescein isothiocyanate;

PE,

phycoerythrin;

PI,

propidium iodide;

FACS,

fluorescence-activated cell sorter