Serum-free coculture system for ex vivo expansion of human cord blood primitive progenitors and SCID mouse-reconstituting cells using human bone marrow primary stromal cells.
OBJECTIVE
In an attempt to maintain and expand human stem cells, many investigators have used xenogeneic, especially murine, stromal cells and fetal calf serum. Because of the possible transmission of infectious diseases, however, the safety of the delivery of grafts expanded in culture using xenogeneic cells and serum has been debated. Using primary human marrow stromal cells, we established a novel serum-free culture system to expand human primitive progenitors and transplantable stem cells.


METHODS
Cord blood CD34(+) cells were cultured on a monolayer of human primary marrow stromal cells in the presence of thrombopoietin (TPO), flt3/flk2 ligand (FL), and/or stem cell factor (SCF) under serum-free conditions. After 2 or 4 weeks of culture, cells were examined for clonogenic progenitors and severe combined immunodeficient disorder (SCID) mouse-reconstituting cells (SRC).


RESULTS
In the presence of TPO, FL, and SCF, marrow stromal cells supported more than a 100- and 1,000-fold expansion of CD34(+) cells and colony-forming units in culture after 2 and 4 weeks of incubation, respectively. In addition, cobblestone area-forming cells were expanded more than 18- and 60-fold after 2 and 4 weeks of culture, respectively. Furthermore, SRC assay demonstrated augmented engraftment by cultured cells.


CONCLUSIONS
This ex vivo expansion system should prove valuable in clinical settings in which stromal cells are available from recipients or stem cell donors.
Yamaguchi
M
M
Hokkaido Red Cross Blood Center, Sapporo, Japan.
Hirayama
F
F
Kanai
M
M
Sato
N
N
Fukazawa
K
K
Yamashita
K
K
Sawada
K
K
Koike
T
T
Kuwabara
M
M
Ikeda
H
H
Ikebuchi
K
K
D000208:Acute Disease; D000818:Animals; D018952:Antigens, CD34 / Q000032:analysis; D001854:Bone Marrow Cells / Q000166:cytology*; D018920:Coculture Techniques / Q000379:methods*; D003114:Colony-Forming Units Assay; D016895:Culture Media, Serum-Free; D005312:Fetal Blood / Q000166:cytology*; D005434:Flow Cytometry; D006410:Hematopoiesis; D018380:Hematopoietic Stem Cell Transplantation; D006412:Hematopoietic Stem Cells / Q000166:cytology*; D006801:Humans; D007938:Leukemia / Q000473:pathology; D017493:Leukocyte Common Antigens / Q000032:analysis; D008565:Membrane Proteins / Q000494:pharmacology; D051379:Mice; D016688:Mice, Inbred NOD; D016513:Mice, SCID; D019089:Stem Cell Factor / Q000494:pharmacology; D017154:Stromal Cells / Q000166:cytology*; D013926:Thrombopoietin / Q000494:pharmacology
D018952:Antigens, CD34; D016895:Culture Media, Serum-Free; D008565:Membrane Proteins; D019089:Stem Cell Factor; C084881:flt3 ligand protein; D013926:Thrombopoietin; D017493:Leukocyte Common Antigens