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Blood, Vol. 93 No. 11 (June 1), 1999:
pp. 3736-3749
Engraftment in Nonobese Diabetic Severe Combined Immunodeficient
Mice of Human CD34+ Cord Blood Cells After Ex Vivo
Expansion: Evidence for the Amplification and Self-Renewal of
Repopulating Stem Cells
Wanda Piacibello,
Fiorella Sanavio,
Antonella Severino,
Alessandra Danè,
Loretta Gammaitoni,
Franca Fagioli,
Eliana Perissinotto,
Giuliana Cavalloni,
Orit Kollet,
Tsvee Lapidot, and
Massimo Aglietta
From the Department of Biomedical Sciences and Human Oncology,
University of Torino Medical School, Torino; the Hematology/Oncology
Section, Mauriziano Hospital, Torino; the Pediatric Department; the
Institute for Cancer Research and Treatment (IRCC), Candiolo, Torino,
Italy; and the Department of Immunology, the Weizmann Institute of
Science, Rehovot, Israel.
Understanding the repopulating characteristics of human
hematopoietic stem/progenitor cells is crucial for predicting their performance after transplant into patients receiving high-dose radiochemotherapy. We have previously reported that CD34+
cord blood (CB) cells can be expanded in vitro for several months in
serum containing culture conditions. The use of combinations of
recombinant early acting growth factors and the absence of stroma was
essential in determining this phenomenon. However, the effect of these
manipulations on in vivo repopulating hematopoietic cells is not known.
Recently, a new approach has been developed to establish an in vivo
model for human primitive hematopoietic precursors by transplanting
human hematopoietic cells into sublethally irradiated nonobese diabetic
severe combined immunodeficient (NOD/SCID) mice. We have examined here
the expansion of cells, CD34+ and
CD34+38 subpopulations, colony-forming
cells (CFC), long-term culture initiating cells (LTC-IC) and the
maintenance or the expansion of SCID-repopulating cells (SRC) during
stroma-free suspension cultures of human CD34+ CB cells
for up to 12 weeks. Groups of sublethally irradiated NOD/SCID mice were
injected with either 35,000, 20,000, and 10,000 unmanipulated
CD34+ CB cells, which were cryopreserved at the start of
cultures, or the cryopreserved cells expanded from 35,000, 20,000, or
10,000 CD34+ cells for 4, 8, and 12 weeks in the presence
of a combination of early acting recombinant growth factors (flt 3/flk2
ligand [FL] + megakaryocyte growth and development factor [MGDF] ± stem cell factor [SCF] ± interleukin-6 [IL-6]).
Mice that had been injected with  20,000 fresh or cryopreserved
uncultured CD34+ cells did not show any sign or showed
little engraftment in a limited number of animals. Conversely, cells
that had been generated by the same number of initial
CD34+ CB cells in 4 to 10 weeks of expansion cultures
engrafted the vast majority of NOD/SCID mice. The level of engraftment,
well above that usually observed when the same numbers of uncultured cells were injected in the same recipients (even in the presence of
irradiated CD34 cells) suggested that primitive
hematopoietic cells were maintained for up to 10 weeks of cultures. In
addition, dilution experiments suggest that SRC are expanded more than
70-fold after 9 to 10 weeks of expansion. These results support and
extend our previous findings that CD34+ CB stem cells
(identified as LTC-IC) could indeed be grown and expanded in vitro for
an extremely long period of time. Such information may be essential to
design efficient stem cell expansion procedures for clinical use.
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