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.

MATERIALS AND 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.

CONCLUSION

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.