Background : It is suggested that clinical practice in the areas of bone marrow
transplantation and gene therapy might rely on the ex vivo expansion of hematopoietic
stenuprogenitor cells. However, the condition for ex vivo expansion of hematopoietic
progenitor cells is not well established. The authors pursued a series of experiments to
define the proper conditions for the expansion of hematopoietic cells in the short-term
liquid suspension culture of bone marrow cells.
Methods :
1.0mL cultures were initiated with 9×10 5 bone marrow
mononuclear cells(BM MNCs) and 9×10³ CD34+ cells, which were isolated
from BM MNCs by high-gradient magnetic cell sorting, in 12 well plates with the
various combinations of heruatopoietic growth factors(HGF). The following recombinant
human HGFs were used: stem cell factor(SCF) l00ng/mL, granulocyte colony-stimulating
factor(G-CSF) l00ng/mL, GM-CSF(granulocyte, macrophage colony-stimulating factor)
l00ng/mL, interieukin-1 beta(IL-1β) 1ng/mL, interleukin-3 (IL-3) 20ng/mL,
interleukin-6(IL-6) 100ng/mL. At the end of culture, colony-forming cells were evaluated
by semisolid clonogenic assay and CD34+ cells were enumerated with flow
cytometry .
Results :
1) In 7-day culture of BM MNCs, colony-forming unit-granulocyte, macrophage(CFU-
GM) numbers expanded 2.8 ∼fold(range, 1.5∼ to 4.2∼fold) with the addition of SCF
alone, 6.3 ∼fold(range, 2.9∼to 13.6∼fold) with SCF plus G-CSF, and 8.8 ∼fold(range,
6.1∼ to 25.1∼fold) with the combination of SCF, G-CSF, IL-1β, and IL-3, respectively.
CD34+cells expanded 4.5 ∼fold(range, 4.0∼ to 5.3-fold) with SCF alone,
5.0∼fold(range, 3.7∼ to 9.1∼fold) with SCFplus G-CSF, and 8.7∼fold(range, 6.9∼ to
10.0∼fold) with the combination of SCF, G-CSF, IL-1β, and IL-3, respectively.
2) Using the high-gradient magnetic sorting system, CD34+ cells were
isolated for an overall of 1.7% of starting MNCs with a yield of 82%.
3) In 7∼day culture of CD34+ cells, CFU-GM numbers expanded 31∼
fold(range, 26∼ to 35∼fold) with the addition of SCF plus G-CSF plus GM-CSF, 40∼
fold(range, 32∼ to 47∼fold) with the combination of SCF, G-CSF, IL-lβ, and IL-3, and
52∼fold(range, 40∼ to 61∼fold) with the combination of all six growth factors. These
results indicate that the expansion of CD34-selected cells is more efficient for the
enrichment of CFU-GM than bone marrow mononuclear cells.
4) Seven-day culture of CD34+ cells was highly superior to 5 ∼day
culture for CFU-GM expansion. 10∼day culture, however, failed to show further
expansion of CFU-GM. Erythroid and multilineage colonies markedly decreased in
number on day 5 of culture and were not observed on day 7 and day 10 of culture.
Conclusion: The authors could confirm that short-term suspension culture of bone
marrow cells could expand hematopoietic progenitor cells. Seven-day culture of
CD34+ cells with the addition of six growth factors, i.e. SCF, G-CSF, IL-1
β, IL-3, IL-6, and GM-CSF, might be the most efficient in this system. To expand
erythroid and multilineage progenitors as well as myeloid progenitors, erythropoietin
seems to be required in short-term culture system.

Keywords: Hematopoietic progenitor cell, Ex vivo expansion, Short-term liquid culture