Preclinical ex vivo expansion of G-CSF-mobilized peripheral blood stem cells: effects of serum-free media, cytokine combinations and chemotherapy.
OBJECTIVE
Ex vivo expansion of granulocyte-colony stimulating factor (G-CSF)-mobilized peripheral blood stem cells (PBSC) is a promising approach for overcoming the developmental delay of bone marrow (BM) reconstitution after transplantation. This project investigated the effects of culture duration, serum-free media, cytokine combinations, and chemotherapy on the outcomes of expansion.


METHODS
Enriched CD34+ cells were cultured for 8 or 10 d in serum-free media (QBSF-60 or X-Vivo 10) and four combinations of cytokines consisting of recombinant human pegylated-megakaryocyte growth and development factor, stem cell factor, flt-3 ligand, G-CSF, interleukin (IL)-6, platelet-derived growth factor (PDGF), and IL-1beta.


RESULTS
Eight days of culture in QBSF-60 significantly supported efficient expansions of CD34+ cells, CD34+ CD38- cells, colony-forming units (CFU) of myeloid, erythroid, megakaryocytic, and mixed lineages to 3.76-, 14.4-, 28.3-, 24.0-, 38.1-, and 15.7-fold, respectively. Whilst PDGF or IL-6 enhanced the expansion of early, myeloid, and erythroid progenitors, IL-1beta specifically promoted the megakaryocytic lineage. Engraftment of human CD45+ cells were detectable in all non-obese diabetic/severe-combined immunodeficient mice transplanted with expanded PBSC from donor samples, being 5.80 +/- 3.34% of mouse BM cells. The expansion and engraftment capacity of CD34+ cells from subjects postchemotherapy were significantly compromised across the panel of progenitor cells.


CONCLUSIONS
Our results provided an optimized protocol for PBSC expansion, applicable to ameliorating neutropenia and thrombocytopenia in post-BM transplant patients by the prompt provision of progenitor cells. For postchemotherapy patients, expansion products might provide committed progenitors for improving short-term engraftment, but not self-renewable stem cells.
Li
Karen
K
Department of Paediatrics, Prince of Wales Hospital, The Chinese University of Hong Kong, Hong Kong, China. lipang@cuhk.edu.hk
Li
Chi Kong
CK
Chuen
Carmen Ka Yee
CK
Tsang
Kam Sze
KS
Fok
Tai Fai
TF
James
Anthony Edward
AE
Lee
Shuk Man
SM
Shing
Matthew Ming Kong
MM
Chik
Kai Wai
KW
Yuen
Patrick Man Pan
PM
D036541:ADP-ribosyl Cyclase / Q000378:metabolism; D051997:ADP-ribosyl Cyclase 1; D000293:Adolescent; D000328:Adult; D015703:Antigens, CD / Q000378:metabolism; D018952:Antigens, CD34 / Q000378:metabolism; D001853:Bone Marrow / Q000473:pathology / Q000502:physiology; D016026:Bone Marrow Transplantation; D049109:Cell Proliferation / Q000187:drug effects*; D002478:Cells, Cultured; D002648:Child; D016895:Culture Media, Serum-Free; D016207:Cytokines / Q000494:pharmacology*; D016179:Granulocyte Colony-Stimulating Factor / Q000494:pharmacology*; D006801:Humans; D008562:Membrane Glycoproteins; D008875:Middle Aged; D023461:Myeloid Progenitor Cells / Q000378:metabolism; D009369:Neoplasms / Q000473:pathology / Q000628:therapy; D013234:Stem Cells / Q000187:drug effects
D015703:Antigens, CD; D018952:Antigens, CD34; D016895:Culture Media, Serum-Free; D016207:Cytokines; D008562:Membrane Glycoproteins; D016179:Granulocyte Colony-Stimulating Factor; D036541:ADP-ribosyl Cyclase; C498154:CD38 protein, human; D051997:ADP-ribosyl Cyclase 1