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Blood, 1 June 2001, Vol. 97, No. 11, pp. 3441-3449
HEMATOPOIESIS
Umbilical cord blood cells capable of engrafting in primary,
secondary, and tertiary xenogeneic hosts are preserved after ex vivo
culture in a noncontact system
Ian D. Lewis,
Graca Almeida-Porada,
Jingbo Du,
Ihor R. Lemischka,
Kateri A. Moore,
Esmail D. Zanjani, and
Catherine M. Verfaillie
From the Stem Cell Institute, the Department of
Medicine and Cancer Center, University of Minnesota, Minneapolis; the
Department of Medicine, the Department of Veterans Affairs Medical
Center, University of Nevada, Reno; and the Department of Molecular
Biology, Princeton University, Princeton, NJ.
This report describes stroma-based and stroma-free cultures that
maintain long-term engrafting hematopoietic cells for at least 14 days
ex vivo. Umbilical cord blood (UCB) CD34+ cells were
cultured in transwells above AFT024 feeders with
fetal-liver-tyrosine-kinase (FL) + stem cell factor (SCF) + interleukin 7 (IL-7), or FL + thrombopoietin (Tpo).
CD34+ progeny were transplanted into nonobese
diabetic-severe combined immunodeficiency (NOD-SCID) mice or preimmune
fetal sheep. SCID repopulating cells (SRC) with multilineage
differentiation potential were maintained in FL-SCF-IL-7 or FL-Tpo
containing cultures for up to 28 days. Marrow from mice highly
engrafted with uncultured or expanded cells induced multilineage human
hematopoiesis in 50% of secondary but not tertiary recipients. Day 7 expanded cells engrafted primary, secondary, and tertiary fetal sheep.
Day 14 expanded cells, although engrafting primary and to a lesser
degree secondary fetal sheep, failed to engraft tertiary recipients. SRC that can be transferred to secondary recipients were maintained for
at least 14 days in medium containing glycosaminoglycans and cytokines
found in stromal supernatants. This is the first demonstration that ex
vivo culture in stroma-noncontact and stroma-free cultures maintains
"long-term" engrafting cells, defined by their capacity to engraft
secondary or tertiary hosts.
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