Characterization and transplantation of induced megakaryocytes from hematopoietic stem cells for rapid platelet recovery by a two-step serum-free procedure.
OBJECTIVE
A complete process for mass generation of megakaryocytes from hematopoietic stem cells under serum-free conditions has great clinical potential for rapid platelet reconstruction in thrombocytopenia patients. We have previously reported on the generation of an optimized serum-free medium (serum-free hematopoietic stem cell medium) for ex vivo expansion of CD34(+) cells. Here, we further generated large amounts of functional megakaryocytes from serum-free expanded CD34(+) cells under a complete and optimal serum-free condition for complying with clinical regulations.


METHODS
Serum substitutes and cytokines were screened and optimized for their concentration for megakaryocyte generation by systemically methods. Serum-free induced megakaryocytes were characterized by surface antigens, gene expression, ex vivo megakaryocyte activation ability, and ability of megakaryocyte and platelet recovery in nonobese diabetic/severe combined immunodeficient mice.


RESULTS
The optimal serum-free megakaryocyte induction medium was Iscove's modified Dulbecco's medium containing serum substitutes (i.e., human serum albumin, human insulin, and human transferrin) and a cytokine cocktail (i.e., thrombopoietin, stem cell factor, Fms-like tyrosine kinase 3 ligand, interleukin-3, interleukin-6, interleukin-9, and granulocyte-macrophage colony-stimulating factor). After induction, induced megakaryocytes expressed CD41a and CD61 surface antigens, nuclear factor erythroid-derived 2 and GATA-1 transcription factors and megakaryocyte activation ability. Importantly, transplantation of induced megakaryocytes could accelerate megakaryocyte and platelet recovery in irradiated nonobese diabetic/severe combined immunodeficient mice.


CONCLUSIONS
In conclusion, we have developed a serum-free megakaryocyte induction medium, and the combination of serum-free megakaryocyte and serum-free hematopoietic stem cell media can generate a large amount of functional megakaryocytes efficiently. Our method represents a promising source of megakaryocytes and platelets for future cell therapy.
Chen
Te-Wei
TW
Bioresource Collection and Research Center, Food Industry Research and Development Institute, Hsinchu, Taiwan.
Hwang
Shiaw-Min
SM
Chu
I-Ming
IM
Hsu
Shu-Ching
SC
Hsieh
Tzu-Bou
TB
Yao
Chao-Ling
CL
D000818:Animals; D018952:Antigens, CD34 / Q000032:analysis; D001773:Blood Cells / Q000166:cytology; D001792:Blood Platelets / Q000166:cytology*; D001854:Bone Marrow Cells / Q000166:cytology; D018929:Cell Culture Techniques / Q000379:methods*; D003470:Culture Media / Q000494:pharmacology; D016895:Culture Media, Serum-Free / Q000494:pharmacology*; D016207:Cytokines / Q000494:pharmacology; D005312:Fetal Blood / Q000166:cytology; D006085:Graft Survival; D006412:Hematopoietic Stem Cells / Q000166:cytology*; D006801:Humans; D039661:Integrin beta3 / Q000096:biosynthesis; D008533:Megakaryocytes / Q000166:cytology / Q000187:drug effects / Q000637:transplantation*; D051379:Mice; D016688:Mice, Inbred NOD; D016513:Mice, SCID; D019038:Platelet Glycoprotein GPIb-IX Complex / Q000096:biosynthesis; D011828:Radiation Chimera; D034061:Thrombopoiesis
D018952:Antigens, CD34; D003470:Culture Media; D016895:Culture Media, Serum-Free; D016207:Cytokines; C061248:ITGB3 protein, human; D039661:Integrin beta3; D019038:Platelet Glycoprotein GPIb-IX Complex