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

There is a large difference between cells isolated from BM and those from CB. In vitro culture of BM CD34⁺ cells results in small percentage (1%) of cells becoming large megakaryocytes by morphology. However for cord blood, there is a significant delay with no large cells appearing at all in the first 12 days of culture. This delay is of significant importance as it introduces many platelet-derived issues during CB transplantations, which are frequently associated with a late recovery and engraftment of peripheral blood cells of donor origin as compared to transplantation of BM or mobilized PB. As a result, patients present with thrombocytopenia and delayed hospital stays due to insufficient platelet counts. To date, the major proponent used in the development and maturation of megakaryocytes is thrombopoietin (TPO) which was used in all our experiments as the control.

We initially discovered 616452 when CD34⁺ cells induced with it produced a significant number of large megakaryocyte-like cells. Within four days of induction, there was an abundance of megakaryocytes, and they only continued to increase in size. These large cells were 2–16 times the size of the typical cells in the control. In fact, the control cells when induced with only TPO had little to no observable increase in cell sizes. Cell cultures typically lasted only eight days after induction due to aggregation and formation of MK-burst forming colonies (Fig. 4i–l). Analysis of cells after day 8 resulted in inconsistent results due to the nature of colonies as well as ease in which the MKs could be lysed by simple pipetting techniques.

These large cells were identified by their morphology, phenotype, and functionality. A Giemsa-Wright stain displayed typical megakaryocyte morphology of lobular and multi-nucleated nucleus with a granular cytoplasm. Analysis by flow and live cell staining revealed these large cells to be CD41 positive, a protein typically expressed on platelets and megakaryocytes. Additionally, the induced cells were found to have drastically increased nuclear DNA content compared to control with cells developing ploidy numbers as great as 64 N within 8 days of induction.

Between experiments (5), there was an inconsistency in the number of large cells among the samples from different patients. While the CD41 population appeared to increase regardless of the patient, the population seemed to differ drastically between certain patients. This was likely caused by the variation of megakaryocyte precursors among patients. Using flow cytometry, we sorted the cells for CD41⁺ and CD41^? cells. Both populations were then induced using the small chemical and TPO. We found that the CD41⁺ population induced with the chemical had increased ploidy of 4 N or greater in over 50% of the cells while the control cells only had approximately 6%, an increase of nearly 10-fold. In the CD41^? population, there was only an increase in 3% of cells of 4 N or greater ploidy compared to non-induced. While not perfect, a small fraction of CD41⁺ cells or hematopoietic stem cell remained after sorting that developed into the MK lineage explains the slight increase in the number of ploidy cells.

Often associated with megakaryopoiesis, one concern was the dependence of this chemical on the presence of or priming by, in the case of CD41⁺ cells, TPO. CB CD34⁺ cells were induced with the chemical for 8 days in the presence and absence of TPO, and it was found that the increase of MK maturation increased in nearly all conditions, with or without TPO (Additional file 2: Figure S2g). However, the presence of cytokines does increase overall cell counts and copy number of MK genes, which produces larger numbers of MKs.

The hematopoietic stem cells (HSCs) of various other species such as monkey and mouse were isolated and induced with the same chemical. A comparison against human HSCs revealed the chemical to be the most potent in monkey with a 30–200% increase in ploidy development of certain sizes and nearly 2000%-fold greater than that of mouse HSCs (Additional file 3: Figure S3). Even so, each species still experienced an increase in ploidy development.

The efficacy of this small molecule on platelet production was initially tested in various mouse models to no avail. It was found that the delivery of this chemical was of great importance. When given via intraperitoneal (IP) injections, the mice experienced no effect and in fact had a negative impact on the mice. The optimal mode of delivery was via intravenous (IV) injections. Many mice models failed initially, but after the discovery that mice HSCs are significantly less impacted by the chemical than that of human or monkey HSCs, a new transplant model was developed. The mice were given a sublethal dose of irradiation at 2.25 Gy to make room for engraftment and then injected with 50000 CD34⁺ cells and 300000 progenitor cells (CD34⁺ cells cultured for 4 days). The mice were then given 3 days of rest to allow the cells to hone into their respective niches after which each mouse was administered one daily dose of the chemical at 15 mg/kg. On day 8, we found that human platelet production in the induced mice were 50% greater than the control mice.

The concentration of 616452 used varied from 2.5–40 uM. Although there appeared to be potent effects at higher concentrations, they could be difficult to achieve in vivo. The side effect profile could be of great concern as mice injected with 50 mg/kg showed significant toxicity (data not shown) with the majority of the mice dying before the conclusion of the in vivo assay. This side effect profile is of great concern when thinking about the translational effect to clinical medicine, and therefore further studies on the toxicities and delivery mechanisms by which to reduce such toxicities are necessary.