Identification of a potent small molecule capable of regulating polyploidization, megakaryocyte maturation, and platelet production
© The Author(s). 2016
Received: 6 September 2016
Accepted: 11 November 2016
Published: 8 December 2016
Megakaryocytic 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.
Human 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.
Within 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.
Our studies may have an important impact on our fundamental understanding of fetal MK biology, the clinical management of thrombocytopenic neonates and leukemic differentiation therapy.
Megakaryocytes are one of few cell types that undergo endomitosis, a form of cell cycle that skips the late stages of mitosis to become polyploid [1–4]. Human megakaryocytes commonly reach ploidy states of 16 N and can achieve states as high as 128 N. The mechanism of polyploidization is still not well understood, however, polyploidy is required for functional human megakaryocyte maturation. Once active, the megakaryocytes are responsible for the production of platelets that have well-characterized roles in hemostasis, thrombosis, vascular integrity, development of the lymphatic system, and the innate immune response [5–8].
Thrombocytopenia affects approximately 20–35% of infants admitted to the neonatal intensive care unit [9–11]. Approximately 9% of those infants are severe and experience clinically significant bleeding (usually intracranial). Platelet transfusions are one of the only therapeutic options for thrombocytopenic neonates. Recent studies have shown that megakaryocytes of neonates are smaller and have lower ploidy than those of adults [12, 13]. Small megakaryocytes usually produce fewer platelets than large megakaryocytes and typically achieve adult size at approximately 1 year of age. Therefore, an inability to increase megakaryocyte size and ploidy in response to increased platelet consumption might underlie the predisposition of sick neonates to thrombocytopenia.
In adults, clinically significant thrombocytopenia is often multifactorial often involving cytotoxic or suppressive effects of chemotherapeutic agents and malignant cells, respectively. Thrombopoietin (TPO) is synthesized in the liver and is the primary regulator of megakaryocyte development and maturation [14, 15]. Recombinant human TPO (rhTPO) has been shown to attenuate carboplatin-induced thrombocytopenia, reducing the need for platelet transfusions . However, the clinical development of rhTPO has since been halted due to the natural development of anti-TPO antibodies in patients. Alternative routes to target TPO receptors such as eltrombopag, a non-peptide, small molecule, that have been shown to stimulate megakaryopoiesis of CD34+ cells in patients with multiple myeloma are in the pipelines [17, 18].
Human umbilical cord blood (hUCB) is an important stem cell source for patients who lack other suitable donors. However, slower platelet engraftment is a major drawback of hUBC transplantation. Platelet engraftment takes an average of approximately 50 days for hUBC recipients, versus 20 days for mobilized peripheral blood cells derived from adult donors . Identification of a megakaryocyte maturation inducer or co-transfusion of large numbers of ex vivo generated human megakaryocyte-committed cells with high maturation potential, could provide an alternative method to shorten period of thrombocytopenia . TPO and its derivatives have been used in the treatment of thrombocytopenia in adult but not neonatal patients. However, studies in models using the non-human primate or canine demonstrated that standard post-transplant admiration of TPO could not accelerate platelet reconstitution following autologous bone marrow transplantation (AuBMT ) or allogenic bone marrow transplantation (alloBMT), respectively, in myeloablated hosts [20–23]. TPO stimulates the megakaryocyte formation in vivo, but it does not shorten its maturation time .
Although the cellular and molecular mechanisms underlying the differences of neonatal and adult MKs remain unclear, studies in congenital disorders have begun to elucidate these mechanisms. A transient myeloproliferative disorder with immature MK features (impaired maturation of MKs) is seen exclusively in fetuses and neonates with Down syndrome and GATA1 mutations indicating that thrombopoietin (TPO)-independent pathways may play a critical role in neonatal/fetal MK maturation [22, 24, 25].
In this manuscript, we introduce and characterize a novel chemical that has not yet been implicated in megakaryopoiesis. We found that this chemical molecule selectively increased polyploidization and shortened maturation of cord blood MKs. The size and ploidy of cord blood/adult mobilized peripheral blood megakaryocytes were also dramatically increased in response to this chemical small molecule stimulation with a dose-dependent fashion. The chemical also induced human peripheral platelet production in mice transplanted with human cord blood. The molecular action of this chemical to stimulate the shortened MK maturation may be through TPO-independent pathways.
Isolation of CD34+ cells from mobilized peripheral blood, bone marrow, or umbilical cord blood
Cells were isolated with MACS CD34+ microbead kit following manufacturer protocols. In the isolation of cord blood, cells underwent a lysis phase for 15 min at room temperature using BD PharmLyse prior to undergoing the MACS CD34+ isolation protocol. Percentage of viable CD34+ obtained ranged from 90–98% in purity.
Standard culture of CB cells used StemSpan SFEM (StemCell Technologies) containing 10% FBS (Gibco), 100 units/ml Pen Strep, 100 ng/ml SCF, TPO, and Flt-3 ligand (Peprotech). For expansion of cells targeted for cell sorting, we used the same media containing a different cytokine cocktail of 100 ng/ml SCF, TPO, and 10 ng/ml IL-3.
616452 and 616454 were purchased from Calbiochem. SB431542 was purchased from Tocris. All chemicals were reconstituted according to manufacturers’ instructions. For animal studies, the 616452 was purchased in bulk from BioVision.
Cells were centrifuged for 5 min at 200g at 4 °C. Pellet was resuspended in 70% ethanol for overnight at 4 °C. After which, the cells were washed with PBS and centrifuged for 5 min at 200g at 4 °C. The pellets were resuspended in PI staining buffer containing PBS with 1% Triton X-100, 100 ul of 2 mg/ml PI, and 0.25 ug/ml of deoxyribonuclease (DNAse)-free ribonuclease (RNAse). The following suspension was incubated at room temperature for 30 min prior to analysis.
Flow cytometry analysis
Cells were centrifuged for 5 min at 200g at 4 °C. Cells were resuspended with CD34, 38, and/or 41 conjugated antibody for 30 min on ice. Cells were then washed with 2 ml of PBS and centrifuged for 5 min at 200g at 4 °C. The final pellet was resuspended using 300 ul of 2% formalin. Analysis was run on the FACSCalibur.
Cells were centrifuged for 5 min at 200g at 4 °C and resuspended with 25 ul of CD41-FITC antibody for 30 min on ice. Cells are then washed gently with PBS and centrifuged for 5 min at 200g at 4 °C. Cells are then suspended at a concentration of 106 cells/ml of PBS containing 2% FBS. Cells are sorted via FACSAria.
Animal study: platelet recovery upon injection of chemical
Twelve 10-week-old NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (Jackson Laboratories) male mice were given sublethal irradiation of 2.25 Gy. The following day, mice were given 50000 CD34+ cells and 300000 progenitor cells (CD34+ cells that were cultured for 4 days) via IV injection. Mice were given daily 616452 injections at 15 mg/kg. Mice were bled via submandibular bleeding every 3–4 days, and the collected blood was analyzed using the HemaVet 950 for platelet and cellular composition. The mice were sacrificed on day 12, and their bone marrow was analyzed for CD34 and CD41.
Gene expression profile
Cells were induced using 616452 for 2 and 4 days with the control cells induced with dimethyl sulfoxide (DMSO). Cells were pelleted and flash frozen in liquid nitrogen and analyzed using PAHS-054Z Hematopoiesis array via Qiagen’s service center.
RNA isolation and quantitative real-time polymerase chain reaction
Total RNA was isolated from human umbilical cord blood cells treated with or without 616452 using the AllPrep DNA/RNA Mini Kit (Qiagen Group). According to the manufacturer’s protocol, 1 ug total RNA from each sample was subjected to complementary DNA (cDNA) synthesis using QuantiTect® Reverse Transcription Kit (Qiagen Group). PCR was carried out using Power SYBRR Green PCR Master Mix (Applied Biosystems) and performed on a 7500-real-time PCR system (Applied Biosystems). The data were analyzed using the delta-delta Ct method.
Identification of small molecules that induce polyploidization
Previously, we have shown that the stem cell gene SALL4 is a robust stimulator of expansion of hematopoietic stem/progenitor cells [26–28] and plays a critical role in hematopoietic differentiation. We screened small molecules targeting SALL4 expression using a SALL4 luciferase reporter. One TGF-β pathway inhibitor, 616452, was identified as a strong inducer of SALL4 expression. When cultured with isolated CD34+ cells, we noticed the appearance of large megakaryocyte-like cells.
Induction of megakaryocyte differentiation and maturation in bone marrow (BM) CD34+
Potent agonist of cord blood (CB) megakaryocyte differentiation and maturation from umbilical cord blood
Accumulated evidence demonstrates that neonatal MKs are significantly smaller, of lower ploidy, and produce fewer platelets than MKs from adults. Based on these characteristics, MKs from fetuses and neonates have been considered to be immature compared with adult MKs. We then tested the chemical effects on neonatal MK maturation. Cells isolated from human umbilical CB were cultured in hematopoietic stem cell (HSC) media induced with 616452 and compared DMSO-treated control cells. Larger cells characteristic of developing megakaryocytes appeared after 4 days of culture (Additional file 1: Figure S1a). The quantity and size of cells increased over time. By 8 days, the culture dishes were predominately composed of megakaryocytes with the formation of megakaryocytic clusters beginning. A Giemsa-Wright stain of these cells revealed a lobular multi-nucleated structure and a granulocytic cytoplasm characteristic of megakaryocytes (Additional file 1: Figure S1c). Flow analysis revealed an increase of 44.8% of the CD41+ population as well as an increase in the CD34+ CD41+ population (Additional file 1: Figure S1b) similar to that of bone marrow.
616452 works independently of TPO
TPO has been highly associated with megakaryopoiesis, and one concern was whether this inhibitor is working in conjunction or is reliant on the presence of TPO. In the absence of TPO, there was limited cell growth, but MK development was still present with ploidy increase of 5–8-fold over the control in cells of 8 N or greater ploidy (Additional file 2: Figure S2g). In the presence of TPO, cells of 8 N ploidy increased dramatically more with a 55-fold increase over control. This likely suggests that 616452 is capable of functioning in a TPO-independent manner. However, it also has large synergistic interactions with TPO in accelerating the maturation of MKs.
Time and dose dependence
Chemical induction of CB CD34+ revealed that the longer the cells were induced, the greater the ploidy development. Between 8 and 12 days of induction, the number of 8 N, 16 N, and 32 N cells nearly doubled (Fig. 2a, b), while the control remains almost unchanged. At 10 uM, chemical induces ploidy in CB CD34+ cells compared with control cells treated with TPO. Multiple replicates with CB from different individuals (n > 3) indicated that there is little or no 16 N ploidy in control cells. Cells treated with varying concentrations of chemical indicated dose dependence: 2.5 uM increased the numbers of 8 N ploidy cells by 10-fold compared to control, whereas 40 uM increased ploidy by 87-fold over the same population (Fig. 2c). There is also a time-dependent response with an increase in ploidy for up to 6 days in culture (Fig. 2d).
MK differentiated in the presence of 616452 demonstrated a much robust capacity of MK differentiation than that of MKs cultured with Rominplostin (Amgen)
The extent of CD41+ megakaryocytic population in response to the induction of 616452
Synergistic effect of combined TGF-β inhibition
Gene expression profile
Genes studied under the effect of 616452
616452 induces human platelet production in NSG mice
Results are presented as the mean +/−the standard deviation. Statistical significance was confirmed using unpaired student’s t test, Mann-Whitney test, or analysis of variance.
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.
616452 is a TGF-β inhibitor that provides a novel pathway for the induction of megakaryocytes that do not work like the SRC kinase inhibitors like diMF or SU6656 or any Aurora A Kinase inhibitors. Unlike other compounds, 616452 does not have much in vitro toxicity at the concentrations tested in this manuscript and is able to enhance the differentiation into megakaryocytes and speed their maturation. This small chemical provides a new approach toward megakaryocytic diseases that has the potential for clinical treatments without unwanted side effects of other inhibitors caused by their toxicity to all cells.
The authors would like to thank Kevin Pinz for his technical help.
This study was supported by a grant from NYSTEM.
Availability of data and materials
All relevant data and materials within this work are made available in this manuscript. Any additional information can be made freely available to any scientist on reasonable request.
NH and YM conceived and designed the studies. YM supported financially. CA worked with patients and collected the major of samples used in these studies. ML and HA contributed to the collection and assembly of data. NH, YM, HA, and ML interpreted the data. All authors read and approved the final manuscript.
YM is a founder of iCell Gene Therapeutics LLC and Marrowsource Therapeutics International LLC. The other authors declare that they have no competing interests.
Consent for publication
Ethics approval and consent to participate
The manuscript involved the use of human and animal samples. All animal procedures and handling of samples are compliant with our institutional IACUC protocol [278623-33] SALL4 in Hematopoiesis and Leukemogenesis, IACUC #: 2001-1771-FAR-11.16.18- MI. All human samples were obtained with informed consent and compliant with IRB protocol: [179617-8] Stem Cells in Hematopoiesis. The assurance number is FWA 00000125.
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