Systematic delineation of optimal cytokine concentrations to expand hematopoietic stem/progenitor cells in co-culture with mesenchymal stem cells.
The major obstacle to the widespread use of umbilical cord blood (UCB) in hematopoietic stem/progenitor (HSC) cell therapy is the low cell dose available. A cytokine cocktail for the ex vivo expansion of UCB HSC, in co-culture with a bone marrow (BM) mesenchymal stem cells (MSC)-derived stromal layer was optimized using an experimental design approach. Proliferation of total cells (TNC), stem/progenitor cells (CD34(+)) and colony-forming units (CFU) was assessed after 7 days in culture, while sole and interactive effects of each cytokine on HSC expansion were statistically determined using a two-level Face-Centered Cube Design. The optimal cytokine cocktail obtained for HSC-MSC co-cultures was composed by SCF, Flt-3L and TPO (60, 55 and 50 ng mL(-1), respectively), resulting in 33-fold expansion in TNC, 17-fold in CD34(+) cells, 3-fold in CD34(+)CD90(+) cells and 21-fold in CFU-MIX. More importantly, these short-term expanded cells preserved their telomere length and extensively generated cobblestone area-forming cells (CAFCs) in vitro. The statistical tools used herein contributed for the rational delineation of the cytokine concentration range, in a cost-effective way, while systematically addressing complex cytokine-to-cytokine interactions, for the efficient HSC expansion towards the generation of clinically significant cell numbers for transplantation.
Andrade
Pedro Z
PZ
IBB-Institute for Biotechnology and Bioengineering, Centre for Biological and Chemical Engineering, Instituto Superior Técnico, Lisboa, Portugal.
dos Santos
Francisco
F
Almeida-Porada
Graça
G
da Silva
Cláudia Lobato
CL
S Cabral
Joaquim M S
JM
D018952:Antigens, CD34 / Q000032:analysis; D001854:Bone Marrow Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D018929:Cell Culture Techniques; D049109:Cell Proliferation / Q000187:drug effects*; D002478:Cells, Cultured; D018920:Coculture Techniques; D003114:Colony-Forming Units Assay; D016207:Cytokines / Q000494:pharmacology*; D005260:Female; D005312:Fetal Blood / Q000166:cytology / Q000378:metabolism; D018380:Hematopoietic Stem Cell Transplantation / Q000379:methods; D006412:Hematopoietic Stem Cells / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D006801:Humans; D008565:Membrane Proteins / Q000494:pharmacology; D059630:Mesenchymal Stem Cells / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D019089:Stem Cell Factor / Q000494:pharmacology; D013926:Thrombopoietin / Q000494:pharmacology; D013997:Time Factors
D018952:Antigens, CD34; D016207:Cytokines; D008565:Membrane Proteins; D019089:Stem Cell Factor; C084881:flt3 ligand protein; D013926:Thrombopoietin