Role of different medium and growth factors on placental blood stem cell expansion: an in vitro and in vivo study.
Expansion of haemopoietic stem cells from placental blood has been obtained with a combination of flt3 ligand (FL), thrombopoietin (TPO), kit-ligand (KL) with or without interleukin-6 (IL6) in serum-replete medium. For clinical use, cell expansion in the absence of serum is a clear advantage. Therefore, stem cell expansion in serum-free (SF) medium with a combination of three (FL, TPO, KL) or four (FL, TPO, KL, IL6) growth factors was compared with the results obtained using fetal calf serum (FCS) or human serum (HS). Human CD34(+) placental blood cells were cultured in the presence of FL, TPO, KL +/- IL6 with SF medium, HS and FCS for up to 8 weeks. CD34(+), CFC, LTC-IC content was measured at intervals. To determine the in vivo repopulating capacity of expanded cells, CD34(+) expanded cells were transplanted in sublethally irradiated NOD/SCID mice. With the three growth factor combination the CD34(+) cell number increased steadily up to the 8 weeks of culture. CD34(+) cells were expanded 67.5-fold with SF, 11.7 with HS and 49.2 with FCS. However, when CFCs and LTC-ICs were considered, a continuous expansion was observed only with HS and FCS, whereas in SF medium after 6 weeks their number started to decline. The addition of IL-6 did not change the expansion significantly. Cells grown ex vivo for 14 days were transplanted into NOD/SCID mice. The engraftment of human cells in mice was higher for serum-replete than for SF expanded cells. Nevertheless, SF cultured cells were also able to engraft both marrow and spleen in all animals. In addition, engrafted human cells still maintained clonogenic ability. With KL, FL, TPO +/- IL6 it is possible to expand haemopoietic progenitor cells in a SF medium. Compared with serum-replete cultures, the absolute number of clonogenic cells and in vivo repopulating cells is lower. Although the degree of expansion remains significant, a clinical trial still needs to be carried out to address the question of whether this expansion might be useful in reducing post-transplant aplasia.
Berger
M
M
Department of Paediatrics, University of Turin Medical School, Turin, Italy.
Fagioli
F
F
Piacibello
W
W
Sanavio
F
F
Mareschi
K
K
Biasin
E
E
Bruno
S
S
Gammaitoni
L
L
Gunetti
M
M
Nesi
F
F
Madon
E
E
Aglietta
M
M
D000818:Animals; D018952:Antigens, CD34 / Q000032:analysis; D001769:Blood; D002417:Cattle; D018929:Cell Culture Techniques / Q000379:methods*; D002455:Cell Division; D003470:Culture Media / Q000494:pharmacology*; D006085:Graft Survival; D006133:Growth Substances / Q000494:pharmacology*; D006412:Hematopoietic Stem Cells / Q000166:cytology* / Q000187:drug effects; D006801:Humans; D051379:Mice; D016513:Mice, SCID; D010920:Placenta; D033581:Stem Cell Transplantation; D014183:Transplantation, Heterologous
D018952:Antigens, CD34; D003470:Culture Media; D006133:Growth Substances