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Ex Vivo Culture of
CD34+/Lin /DR Cells in
Stroma-Derived Soluble Factors, Interleukin-3, and Macrophage
Inflammatory Protein-1 Maintains Not Only Myeloid But
Also Lymphoid Progenitors in a Novel Switch Culture Assay
Jeffrey S. Miller,
Valarie McCullar, and
Catherine M. Verfaillie
From the Department of Medicine, University of Minnesota Cancer
Center, Minneapolis, MN.
We have demonstrated that long-term culture initiating cells
(LTC-IC) are maintained in a stroma noncontact (SNC) culture where
progenitors are separated from stroma by a microporous membrane and
LTC-IC can proliferate if the culture is supplemented with interleukin-3 (IL-3) and macrophage inflammatory protein-1
(MIP-1). We hypothesize that the same conditions, which result in
LTC-IC proliferation, may also maintain lymphoid progenitors. Natural killer (NK) cells are of lymphoid lineage and a stromal-based culture
can induce CD34+/Lin /DR
cells to differentiate along the NK cell lineage. We developed a
three-step switch culture assay that was required to demonstrate the
persistence of NK progenitors in
CD34+/Lin/DR cells assayed
in SNC cultures supplemented with IL-3 and MIP-1. When
CD34+/Lin/DR progeny from
the SNC culture were plated sequentially into "NK cell progenitor
switch" conditions (contact with stromal ligands, hydrocortisone-containing long-term culture medium, IL-2, IL-7, and
stem cell factor [SCF]) followed by "NK cell differentiation" conditions (contact with stromal ligands, human serum, no
hydrocortisone, and IL-2), significant numbers of
CD56+/CD3 NK resulted, which exhibited
cytotoxic activity against K562 targets. All steps are required because
a switch from SNC cultures with IL-3 and MIP-1 directly to "NK
cell differentiation" conditions failed to yield NK cells suggesting
that critical step(s) in lymphoid commitment were missing. Additional
experiments showed that CD34+/CD33 cells
present after SNC cultures with IL-3 and MIP-1, which contained up
to 30% LTC-IC, are capable of NK outgrowth using the three-step switch
culture. Limiting dilution analysis from these experiments showed a
cloning frequency within the cultured CD34+/CD33 population similar to fresh
sorted CD34+/Lin/DR cells.
However, after addition of FLT-3 ligand, the frequency of primitive
progenitors able to develop along the NK lineage increased 10-fold. In
conclusion, culture of primitive adult marrow progenitors ex vivo in
stroma-derived soluble factors, MIP-1, and IL-3 maintains both very
primitive myeloid (LTC-IC) and lymphoid (NK) progenitors and suggests
that these conditions may support expansion of human hematopoietic stem
cells. Addition of FLT-3 ligand to IL-2, IL-7 SCF, and stromal factors
are important in early stages of NK development.
Blood, Vol. 91 No. 12 (June 15), 1998:
pp. 4516-4522
© 1998 by The American Society of Hematology.
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