Ex vivo expansion of human UC blood primitive hematopoietic progenitors and transplantable stem cells using human primary BM stromal cells and human AB serum.
BACKGROUND
In vitro maintenance and expansion of human hematopoietic stem cells is crucial for many clinical applications, and investigators have been using xenogeneic, especially murine, stromal cells for stem-cell expansion. In addition, many such culture systems utilize FCS-containing medium or serum-free medium that contains human- or animal-derived proteins. However, the possible transmission of infectious diseases has led to a debate about the safety of the delivery of grafts expanded in culture using cells and proteins of allogeneic or xenogeneic origin. Using primary human BM stromal cells, we have established an AB serum-based co-culture system to expand human primitive progenitors and transplantable stem cells.


METHODS
Cord blood CD34+ cells were cultured on a monolayer of human BM-derived primary stromal cells with thrombopoietin (TPO), stem-cell factor (SCF) and flt3/flk2 ligand (FL) in the presence of either FCS or AB serum. One to three weeks later, cells were examined for total cells, CD34+ cells, CD34+ CD38- cells, and clonogenic progenitors. SCID mouse reconstituting cell (SRC) activity was also studied.


RESULTS
Three weeks of culture with TPO, SCF, and FL supported more than a 250-fold expansion of CD34+ cells, CD34+ CD38- cells and CFU-C, regardless of the kind of serum used. SRC assay revealed that transplantable stem cells were moderately expanded as well.


CONCLUSIONS
This ex vivo expansion system should prove valuable in clinical settings in which stromal cells and serum are available from recipients or stem-cell donors.
Yamaguchi
M
M
Hokkaido Red Cross Blood Center, Sapporo, Japan.
Hirayama
F
F
Murahashi
H
H
Azuma
H
H
Sato
N
N
Miyazaki
H
H
Fukazawa
K
K
Sawada
K
K
Koike
T
T
Kuwabara
M
M
Ikeda
H
H
Ikebuchi
K
K
D036541:ADP-ribosyl Cyclase / Q000378:metabolism; D051997:ADP-ribosyl Cyclase 1; D000818:Animals; D015703:Antigens, CD / Q000378:metabolism; D018952:Antigens, CD34 / Q000378:metabolism; D018929:Cell Culture Techniques / Q000379:methods*; D002478:Cells, Cultured; D002999:Clone Cells; D018920:Coculture Techniques; D016895:Culture Media, Serum-Free; D005312:Fetal Blood / Q000166:cytology*; D006412:Hematopoietic Stem Cells; D008562:Membrane Glycoproteins; D008565:Membrane Proteins / Q000378:metabolism; D051379:Mice; D016688:Mice, Inbred NOD; D016513:Mice, SCID; D019089:Stem Cell Factor / Q000378:metabolism; D033581:Stem Cell Transplantation; D017154:Stromal Cells; D013926:Thrombopoietin / Q000378:metabolism
D015703:Antigens, CD; D018952:Antigens, CD34; D016895:Culture Media, Serum-Free; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D019089:Stem Cell Factor; C084881:flt3 ligand protein; D013926:Thrombopoietin; D036541:ADP-ribosyl Cyclase; C498155:Cd38 protein, mouse; D051997:ADP-ribosyl Cyclase 1