Bone marrow niche-inspired, multiphase expansion of megakaryocytic progenitors with high polyploidization potential.
OBJECTIVE
Megakaryopoiesis encompasses hematopoietic stem and progenitor cell (HSPC) commitment to the megakaryocytic cell (Mk) lineage, expansion of Mk progenitors and mature Mks, polyploidization and platelet release. pH and pO2 increase from the endosteum to sinuses, and different cytokines are important for various stages of differentiation. We hypothesized that mimicking the changing conditions during Mk differentiation in the bone marrow would facilitate expansion of progenitors that could generate many high-ploidy Mks.


METHODS
CD34+ HSPCs were cultured at pH 7.2 and 5% O2 with stem cell factor (SCF), thrombopoietin (Tpo) and all combinations of Interleukin (IL)-3, IL-6, IL-11 and Flt-3 ligand to promote Mk progenitor expansion. Cells cultured with selected cytokines were shifted to pH 7.4 and 20% O2 to generate mature Mks, and treated with nicotinamide (NIC) to enhance polyploidization.


RESULTS
Using Tpo + SCF + IL-3 + IL-11, we obtained 3.5 CD34+ CD41+ Mk progenitors per input HSPC, while increasing purity from 1% to 17%. Cytokine cocktails with IL-3 yielded more progenitors and mature Mks, although the purities were lower. Mk production was much greater at higher pH and pO2. Although fewer progenitors were present, shifting to 20% O2 /pH 7.4 at day 5 (versus days 7 or 9) yielded the greatest mature Mk production, 14 per input HSPC. NIC more than doubled the percentage of high-ploidy Mks to 40%.


CONCLUSIONS
We obtained extensive Mk progenitor expansion, while ensuring that the progenitors could produce high-ploidy Mks. We anticipate that subsequent optimization of cytokines for mature Mk production and delayed NIC addition will greatly increase high-ploidy Mk production.
Panuganti
Swapna
S
Department of Chemical and Biological Engineering, Northwestern University, Evanston, Illinois 60208, USA.
Papoutsakis
Eleftherios T
ET
Miller
William M
WM
D018952:Antigens, CD34 / Q000096:biosynthesis; D001792:Blood Platelets / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D001853:Bone Marrow / Q000378:metabolism / Q000473:pathology*; D018929:Cell Culture Techniques / Q000379:methods; D002454:Cell Differentiation / Q000187:drug effects / Q000235:genetics; D019070:Cell Lineage; D049109:Cell Proliferation; D002478:Cells, Cultured; D006801:Humans; D007378:Interleukins / Q000378:metabolism; D055016:Megakaryocyte Progenitor Cells / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D009536:Niacinamide / Q000494:pharmacology; D017713:Platelet Transfusion; D011123:Polyploidy; D019089:Stem Cell Factor / Q000378:metabolism; D055153:Stem Cell Niche / Q000378:metabolism / Q000473:pathology*; D013921:Thrombocytopenia / Q000473:pathology / Q000628:therapy*; D013926:Thrombopoietin / Q000378:metabolism
D018952:Antigens, CD34; D007378:Interleukins; D019089:Stem Cell Factor; D009536:Niacinamide; D013926:Thrombopoietin