Identification of a potent small molecule capable of regulating polyploidization, megakaryocyte maturation, and platelet production

Abstract

BackgroundMegakaryocytic cell maturation involves polyploidization, and megakaryocyte (MK) ploidy correlates with their maturation and platelet production. Retardation of MK maturation is closely associated with poor MK engraftment after cord blood transplantation and neonatal thrombocytopenia. Despite the high prevalence of thrombocytopenia in a range of setting that affect infants to adults, there are still very limited modalities of treatment.MethodsHuman CD34+ cells were isolated from cord blood or bone marrow samples acquired from consenting patients. Cells were cultured and induced using 616452 and compared to current drugs on the market such as rominplostim or TPO. Ploidy analysis was completed using propidium iodide staining and flow cytometry analysis. Animal studies consisted of transplanting human CD34+ cells into NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ mice followed by daily injections of 15 mg/kg of 616452.ResultsWithin one week of culture, the chemical was able to induce polyploidization, the process required for megakaryocyte maturation with the accumulation of DNA content, to 64 N or greater to achieve a relative adult size. We observed fold increases as high as 200-fold in cells of 16 N or greater compared to un-induced cells with a dose-dependent manner. In addition, MK differentiated in the presence of 616452 demonstrated a more robust capacity of MK differentiation than that of MKs cultured with rominplostim used for adult idiopathic thrombocytopenic purpura (ITP) patients. In mice transplanted with human cord blood, 616452 strikingly enhanced MK reconstitution in the marrow and human peripheral platelet production. The molecular therapeutic actions for this chemical may be through TPO-independent pathways.ConclusionOur studies may have an important impact on our fundamental understanding of fetal MK biology, the clinical management of thrombocytopenic neonates and leukemic differentiation therapy.Electronic supplementary materialThe online version of this article (doi:10.1186/s13045-016-0358-y) contains supplementary material, which is available to authorized users.

Mesenchymal stem cell deficiency influences megakaryocytopoiesis through the TNFAIP3/NF‐κB/SMAD pathway in patients with immune thrombocytopenia

“…MKs from ITP patients displayed abnormal maturation with normal or higher total counts. Human MKs commonly reach ploidy states of 8–128N, and polyploidy is required for functional human MK maturation (Huang et al , ). Our study investigated the abnormal decreased TNFAIP3 expression that induced the activation of the NF‐κB/SMAD7pathway, leading to dysfunctional MSCs that are unable to support ploidy development and platelet formation in ITP.…”

“…MKs from ITP patients display abnormal maturation with normal or higher total counts. Human MKs commonly reach ploidy states of 8–128N, and polyploidy is required for functional MK maturation (Huang et al , ). ITP MKs may lack granularity, which is shown by reduced ploidy (Cines et al , ).…”

Pan-myeloid Differentiation of Human Cord Blood Derived CD34⁺ Hematopoietic Stem and Progenitor Cells

“…Thus, it can be employed for studying normal myeloid differentiation and for investigating the impact of myeloid disease (myelodysplastic syndromes, acute myeloid leukemia, myeloproliferative neoplasms, and others) associated point mutations and chromosomal translocations on molecular and cellular phenotype during proliferation and differentiation of CD34 + cells 11,12,[23][24][25] . This protocol can also be employed for examining the impact of anti-and pro-inflammatory cytokines, and of potential therapeutic drugs on myeloid differentiation [26][27][28] . The pluripotent hematopoietic stem cell (HSC) differentiates into the multipotent progenitor (MPP), which gives rise to the common myeloid progenitor (CMP) and the common lymphoid progenitor (CLP) cells.…”

RUNX1 haploinsufficiency causes a marked deficiency of megakaryocyte-biased hematopoietic progenitor cells: Mechanistic studies and drug correction

“…RepSox (RS), a small compound TGFβR1 inhibitor, promotes human megakaryopoiesis and increases RUNX1 expression in vitro and in vivo-platelet release in mice 69 . RS-treatment improved iMk yield from L1 -iHPCs to near L1-C levels ( Figure 5C).…”