Preclinical ex vivo expansion of cord blood hematopoietic stem and progenitor cells: duration of culture; the media, serum supplements, and growth factors used; and engraftment in NOD/SCID mice.
BACKGROUND
Ex vivo expansion of cord blood (CB) hematopoietic stem and progenitor cells increases cell dose and may reduce the severity and duration of neutropenia and thrombocytopenia after transplantation. This study's purpose was to establish a clinically applicable culture system by investigating the use of cytokines, serum-free media, and autologous plasma for the expansion of CB cells and the engraftment of expanded product in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice.


METHODS
Enriched CB CD34+ cells were cultured in four media (Iscove's modified Dulbecco's medium with FCS, Gibco; X-Vivo-10, BioWhittaker; QBSF-60, Quality Biological; and StemSpan SFEM, Stem Cell Technologies) with four cytokine combinations (thrombopoietin [TPO], SCF, Flt-3 ligand [FL] with and without G-CSF, and/or IL-6). The effect of autologous CB plasma was also investigated. The read-out measures were evaluated on Days 8 and 12. After expansion at the optimized condition, cultured cells were transplanted into sublethally irradiated NOD/SCID mice. The engraftment of human CD45+ cells and subsets in the bone marrow, spleen, and peripheral blood was determined.


RESULTS
QBSF-60 or StemSpan SFEM supported high yields of early progenitors (CD34+ cells, <or= 64.8-fold; CD34+CD38- cells, 330-fold; CFU-granulocyte erythroid macrophage megakaryocyte [GEMM], 248-fold) and CFUs of the myeloid (CFU-GM, 407-fold) and erythroid (BFU/CFU-E, 144-fold) lineages. The expansion of the megakaryocytic lineage was consistently higher in X-Vivo-10 (CFU-megakaryocyte, 684-fold). Autologous plasma promoted colony formation but reduced CD34+ cells and CFU-GEMM. The addition of G-CSF or IL-6 improved cell yields; G-CSF was more effective for committed progenitors. Expansion products from cultures in QBSF-60 with the cytokines engrafted and differentiated into the myeloid and lymphoid lineages in NOD/SCID mice.


CONCLUSIONS
The data supported the strategy of expansion. The optimized condition may be applicable to clinical expansion for the abrogation or reduction of posttransplant cytopenia.
Lam
A C
AC
Department of Paediatrics, Laboratory Animal Services Centre, Prince of Wales Hospital, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong.
Li
K
K
Zhang
X B
XB
Li
C K
CK
Fok
T F
TF
Chang
A M
AM
James
A E
AE
Tsang
K S
KS
Yuen
P M
PM
D000818:Animals; D018952:Antigens, CD34 / Q000032:analysis; D018929:Cell Culture Techniques / Q000379:methods / Q000592:standards; D019070:Cell Lineage; D003470:Culture Media / Q000494:pharmacology / Q000592:standards; D005312:Fetal Blood / Q000166:cytology*; D006085:Graft Survival; D006133:Growth Substances / Q000494:pharmacology; D018380:Hematopoietic Stem Cell Transplantation; D006412:Hematopoietic Stem Cells / Q000166:cytology*; D006801:Humans; D051379:Mice; D016688:Mice, Inbred NOD; D016513:Mice, SCID; D014183:Transplantation, Heterologous
D018952:Antigens, CD34; D003470:Culture Media; D006133:Growth Substances