Enhanced generation of megakaryocytes from umbilical cord blood-derived CD34(+) cells expanded in the presence of two nutraceuticals, docosahexanoic acid and arachidonic acid, as supplements to the cytokine-containing medium.
OBJECTIVE
Ex vivo generation of megakaryocytes (MK) from hematopoietic stem cells (HSC) is important for both basic research, to understand the mechanism of platelet biogenesis, and clinical infusions, for rapid platelet recovery in thrombocytopenic patients. We investigated the role of two nutraceuticals, docosahexanoic acid (DHA) and arachidonic acid (AA), in the in vitro generation of MK.


METHODS
Umbilical cord blood (UCB)-derived CD34+cells were cultured with stem cell factor (SCF) and thrombopoietin (TPO) in the presence (test) or absence (control) of the two additives. On day 10, MK and platelets generated were quantitated by morphologic, phenotypic and functional assays.


RESULTS
The cell yield of MK and platelet numbers were significantly higher in test compared with control cells. Phenotypic analyzes and gene expression profiles confirmed these findings. Functional properties, such as colony-forming unit (CFU)-MK formation, chemotaxis and platelet activation, were found to be enhanced in cells cultured with nutraceuticals. The engraftment potential of ex vivo-expanded cells was studied in NOD/SCID mice. Mice that received MK cultured in the presence of DHA/AA engrafted better. There was a reduction in apoptosis and total reactive oxygen species (ROS) levels in the CD41(+) compartment of the test compared with control sets. The data suggest that these compounds probably exert their beneficial effect by modulating apoptotic and redox pathways.


CONCLUSIONS
Use of nutraceuticals like DHA and AA may prove to be a useful strategy for efficient generation of MK and platelets from cord blood cells, for future use in clinics and basic research.
Stem Cell Lab, National Center for Cell Science, Ganeshkhind, Pune, Maharashtra, India.
Nikhat Firdaus A
NF
Siddiqui
Namrata C
NC
Shabrani
Vaijayanti P
VP
Kale
Lalita S
LS
Limaye
D000818:Animals; D018952:Antigens, CD34 / Q000378:metabolism*; D017209:Apoptosis / Q000187:drug effects; D016718:Arachidonic Acid / Q000494:pharmacology*; D001792:Blood Platelets / Q000166:cytology / Q000187:drug effects; D002454:Cell Differentiation / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D002633:Chemotaxis / Q000187:drug effects; D003114:Colony-Forming Units Assay; D003470:Culture Media / Q000737:chemistry*; D016207:Cytokines / Q000494:pharmacology*; D019587:Dietary Supplements; D004281:Docosahexaenoic Acids / Q000494:pharmacology*; D005312:Fetal Blood / Q000166:cytology*; D006801:Humans; D008533:Megakaryocytes / Q000166:cytology* / Q000637:transplantation; D051379:Mice; D016513:Mice, SCID; D010641:Phenotype; D015539:Platelet Activation / Q000187:drug effects; D011003:Ploidies; D017382:Reactive Oxygen Species / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism
D018952:Antigens, CD34; D003470:Culture Media; D016207:Cytokines; D017382:Reactive Oxygen Species; D014157:Transcription Factors; D004281:Docosahexaenoic Acids; D016718:Arachidonic Acid