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Abstract

Background:

Eltrombopag (EP) is a small molecule that acts directly on hematopoietic stem cells (HSCs) and megakaryocytes to stimulate the hematopoietic process. Mesenchymal stem/stromal cells (MSCs) are key hematopoietic niche regulators.

Objectives:

We aimed to determine whether EP has any effect on MSC function and properties (especially on their hematopoietic-supporting ability) and if so, what changes (e.g. genome-wide transcriptomic alterations) are induced in MSC after EP treatment.

Design/Methods:

MSCs were isolated from 12 healthy donors and treated with 15 µM and 50 µM of EP for 24 h. The toxicity of the drug on MSCs and their differentiation ability were analyzed, as well as the transcriptomic profile, reactive oxygen species (ROS) and DNA damage and the changes induced in the clonogenic capacity of HSCs.

Results:

The results show that EP also modifies MSC functions, decreasing their adipogenic differentiation, increasing the expression of genes involved in hypoxia and other pathways related to oxygen homeostasis, and enhancing their ability to support hematopoiesis in vitro.

Conclusion:

Our findings support the use of EP in cases where hematopoiesis is defective, despite its well-known direct effects on hematopoietic cells. Our findings suggest that further studies on the effects of EP on MSCs from patients with aplastic anemia are warranted.

Keywords

Aplasia Eltrombopag Hematopoiesis Mesenchymal stem cells MSC

Introduction

Eltrombopag (EP) is a small molecule that binds to the transmembrane region of the thrombopoietin (TPO) receptor c-MPL, activating downstream signaling in hematopoietic stem cells (HSCs) and megakaryoblasts. These include the JAK/STAT, PI3 K/AKT and Ras/MAPK pathways.^1,2 It does not compete directly in its binding to c-MPL with TPO, so TPO and EP signaling effects are both synergic.³ In addition, it has a potent ability to bind iron ions, stimulating hematopoiesis through molecular reprogramming⁴ and expanding the most immature populations during hematopoiesis.⁵

Based on these properties, EP is approved for chronic immune thrombocytopenia (ITP) patients who are refractory to other treatments, thrombocytopenia in patients with chronic hepatitis C and in those with acquired severe aplastic anemia.^6–8

EP significantly affects HSCs that are intimately related to the hematopoietic microenvironment that acts as a key regulator of hematopoiesis. Mesenchymal stem/stromal cells (MSCs) regulate the self-renewal, survival, migration, and differentiation of HSCs through cytokine release, the production of growth factors, cell contact interactions and the formation of extracellular matrix.⁹ Some data suggest that MSCs from patients with bone marrow (BM) disorders may be altered, and, in a previous study by our group, we observed that MSCs from patients with ITP are functionally abnormal, with reduced proliferation and immunomodulation ability.¹⁰ Other changes have also been described in MSCs from these subjects.^11–13 In addition, MSCs may be altered in severe aplastic anemia patients, although the results are controversial.^14–16 These suggested alterations in MSCs from patients with ITP or AA make it even more worthwhile to evaluate the effects of EP on healthy-donor-derived MSCs.

The effects of EP on HSCs are well documented, but its potential action on the microenvironment, especially on MSCs, has not so far been addressed in depth. A recent report has analyzed the effects of EP on BM-derived MSCs from ITP patients, showing that EP can reduce MSC apoptosis and increase their proliferation, interfering with intracellular iron signaling in cells from these patients, whereas the changes observed in control cells in that particular report were minimal.¹⁷ Nevertheless, neither the effects of EP on the hematopoietic supporting ability of MSC, nor the genomic changes induced by the drug has been assessed. These are therefore the main objectives of the current work.

Methods

Reagents

Eltrombopag was provided by Novartis Pharmaceuticals. The drug was dissolved in sterile water at a concentration of 10 mM. MSCs were cultured with EP in a final concentration of (10–50 μM). Control cells were incubated in the same medium without the drug for the same periods.

Patient samples

MSCs were isolated from bone marrow (BM) from 12 healthy donors (8 female/4 male) with a median age of 43.5 years (range: 18–60 years). All healthy donors were selected following the same criteria and evaluations as used with our allogeneic hematopoietic stem cell transplant donors. These followed international standards (FACT-JACIE) and consisted of a clinical and biological evaluation to confirm the absence of comorbidities. The donors signed a specific informed consent form approved by the Ethics Committee of the University Hospital of Salamanca. Finally, at the time of BM aspiration, normal BM cytomorphology was confirmed by an experienced hematologist.

BM was aspirated from the iliac crest in sterile conditions under propofol sedation. Mononuclear cells (MNCs) were obtained after Ficoll-Paque density-gradient centrifugation (1.077 g/ml; GE Health-care BioSciences, AB, Uppsala, Sweden), and seeded at 1 × 10⁶ MNC/cm² on culture flasks with Dulbecco’s Modified Eagle Medium (DMEM) + 10% SBF + 1% penicillin/streptomycin according to previously described methods standardized in our laboratory.¹⁸ MSCs were expanded and sub-cultured until the third passage (MSC-BMp3), and then used for the experimental procedures described below.

Cell viability assays

Cell viability of MSCs was evaluated in triplicate by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) method.¹⁹ Briefly, MSCs were seeded into 96-well culture dishes (8000 cells/well) in the absence (control) or presence of EP at different concentrations (ranging from 10 to 50 µM) for 24 h. Cells were then incubated for 4 h with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Bio-Techne R&D Systems), following the manufacturer’s instructions. Results are expressed as percentages of live cells.

All the subsequent experiments were performed at the selected doses of 15 µM and 50 µM for 24 h, using untreated MSCs as controls.

MSC differentiation ability

Standard multilineage differentiation assays in adipocytic and osteoblastic lineages were performed after culturing MSCs with EP, following the established protocols described in detail²⁰ and summarized below.

Adipocytic differentiation

In all, 2 × 10⁵ MSCs were seeded and treated with or without EP. After 24 h, the drug was removed, the culture was washed twice with phosphate buffered saline (PBS) and the adipocyte induction medium (MesenCult^TM MSC Basal Medium, Stemcell^TM Technologies) was added for 21 days, changing the medium twice a week. Oil Red staining was performed to assess the extent of adipocytic differentiation.

The number of adipocytes per field formed in each condition was scored weekly (10 fields per sample). The mean of each sample was calculated, the results being summarized as medians.

Osteogenic differentiation

In the same way, 3 × 10⁴ MSCs were stimulated with osteogenic differentiation medium (StemMACS Osteodiff Media, Millenyi Biotec) supplemented with 1% penicillin/streptomycin 1000 U/ml, Gibco) for 10 days. The medium was changed every 3 days. As controls, drug-free MSCs were cultured with differentiation medium, and EP-treated or untreated MSCs were cultured without specific induction medium.

Osteoblasts were stained with alkaline phosphatase, for which purpose NBT (4-nitro blue tetrazolium chloride) and BCIT (5-bromo-4-chloro-3-indolyl phosphate) (Roche) were mixed. Subsequently, the cells were counterstained with 1 ml of hematoxylin (Sigma-Aldrich) for 1 min at RT. Images of adipocytes and osteoblasts were captured under an Olympus BX41 inverted microscope connected to a Canon EOS 650D digital camera.

Mineralized bone quantification

Alizarin red staining was performed to evaluate calcium deposits in MSCs after culture with EP and osteogenic differentiation.²¹ After 21 days, the supernatant was removed, and cells were fixed with formaldehyde and stained with alizarin red (40 mM, pH 4.2). Cells were detached with 10% glacial acetic acid and transferred to an Eppendorf tube. Cells were then heated to 85ºC for 10 min and the supernatant was subjected to a colorimetric assay and absorbance was measured in a spectrophotometer at 405 nm.

Reverse transcription real-time PCR analyses of genes involved in MSC differentiation

Total RNA was obtained from MSCs after treatment with EP and differentiated (n = 4) as indicated before using TriPure isolation reagent (Roche Diagnostics; Mainheim, Germany). Reverse transcription was performed using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA), according to the manufacturer’s instructions. The genes were quantified using the Step One Plus Real-Time PCR System and TaqMan® Gene Expression Assays (Applied Biosystems, Foster City, CA, USA). Gene expression was normalized as values of 2^-ΔCt, where ΔCt = Ct_gene–Ct_GADPH. The genes analyzed are shown in Table 1.

Table 1.

Genes studied to confirm adipogenic and osteogenic differentiation.

Cat.#	Description	Name
Hs00371239_m1	AEBP1	Adipocyte enhancer binding protein 1
Hs01547870_m1	HSD11B1	11 beta 1 hydroxysteroide dehydrogenase 1
Hs01115512_m1	PPARg	Peroxisome proliferative activated receptor
Hs00758162-m1	ALPL	Alkaline phosphatase
Hs00231692-m1	RUNX2	RUNX Family Transcription Factor 2
Hs01055564-m1	BMP2	Bone Morphogenetic Protein 2
Hs01587814-g1	BGLAP	Bone Gamma-Carboxyglutamate Protein
Hs002777762-m1	SPARC	Osteonectin
Hs00959010-m1	SPP1	Osteopontin

Gene expression array

Total RNA of MSCs from four donors treated with 0 µM (control), 15 µM or 50 µM of EP were isolated and purified using RNeasy Kit (Qiagen, Valencia, CA, USA). RNA integrity was confirmed by Agilent 2100 Bioanalyzer (Agilent, Palo Alto, CA, USA). In all, 100 ng of total RNA were amplified, labeled using the WT Plus reagent kit (Affymetrix) and then hybridized to Clariom S human Array (Affymetrix). Washing and scanning were carried out using the Affymetrix GeneChip System (GeneChip Hybridization Oven 645, GeneChip Fluidics Station 450, and GeneChip Scanner 7G).

Array analysis compared untreated MSCs versus MSCs treated with 15 µM and 50 µM of EP, respectively. Differential expression analysis was performed with Limma, a package in R, available in Bioconductor software.

To explore the functions and pathways in which these genes are involved, a search in GeneCards²² and a functional enrichment analysis with the bioinformatics tool WebGestalt were performed.

Gene expression analysis by RT-PCR

To confirm the results obtained from the gene expression array, total RNA was extracted from treated and control MSCs (n = 7) using Trizol. Gene expression of BNIP-3 (Hs00969291-m1), VEFGa (Hs00900054-m1), HIF1-α (Hs00153153-m1), BDH-2 (Hs00560373-g1), and GADPH (Hs99999905-m1) as a control gene, was analyzed in the same way as previously described.

Functional assays

To evaluate the functional implications of some of the findings observed in the functional enrichment analysis, the following assays were performed.

Intracellular reactive oxygen species (ROS) levels

MSCs treated with EP and control MSCs were trypsinized, washed, and resuspended in 250 µL of DPBS and 5 µL of 2’-7’-dichlorodihydrofluorescein diacetate 10 µM (H2DCF-DA) (Sigma-Aldrich) for 30 min at 37°C, in the dark and under hypoxic conditions.²³ The fluorescence intensity of 2’,7’-dichlorofluorescein (DCF) was measured in a BD FACS CantoII^TM flow cytometer. Median DCF fluorescence intensity values, directly related to intracellular levels of ROS, were analyzed with Infinicyt^TM Software (Cytognos).

DNA damage analysis

MSCs from both experimental groups were incubated with the Muse^TM Multi-Color DNA Damage Kit (EDM Millipore Corporation) according to the manufacturer’s protocol. Briefly, 1X assay buffer was added to the cells, which were fixed and permeabilized with fixation buffer and permeabilization buffer, respectively. After washing, they were labeled with two antibodies (20X anti-phospho-histone H2A.X-PECy5, 20X anti-phospho-ATM-PE) which measures the phosphorylation status of the histone pH2A.X and the ATM kinase. Cells were acquired with a BD FACS CantoII^TM flow cytometer and results were obtained using the FlowJoTM 10 software (BD Biosciences).

Clonogenic capacity

Mobilized CD34⁺ progenitor cells were isolated from leukapheresis samples from 5 HD (male/female ratio: 3/2; median age: 34 years, range: 18–54 years) as previously described.¹⁸ CD34⁺ progenitor cells were labeled using the human CD34 MicroBead Kit (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) and purified in an AUTOMACs device. The purity and viability of immunomagnetically sorted CD34⁺ cells were determined by flow cytometry using fluorescein isothiocyanate (FITC)-conjugated CD34 (11-0349-42, eBioscience Inc., San Diego, CA, USA) and 7AAD.

These cells were then co-cultured with MSCs pretreated or not with EP. After 24 h of co-culture, 2 × 10³CD34⁺ were seeded in methylcellulose semisolid MACS medium supplemented with stem cell factor (SCF), granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF), IL-3, and IL-6 (Miltenyi Biotec GmbH, Germany) and maintained for 14 days in a humidified atmosphere at 37ºC with 5% CO₂. CFU-GM colonies were then scored with an inverted microscope, as previously described.²⁴

Statistical tests

Statistical analyses were performed with IBM SPSS Statistics v.26 for Windows (IBM Corp., Armonk, NY, USA) using the non-parametric Wilcoxon test to compare the differences between paired results. Graphs were generated with GraphPad Prism 8.4.2 (GraphPad Software Inc., San Diego, CA, USA). Differences were considered statistically significant for values of p < 0.05 (*p < 0.05; **p < 0.01).

Results

Eltrombopag increases the viability of MSCs

The dose–response curves obtained with the MTT assays revealed that EP is not cytotoxic for MSCs obtained from healthy donors. Moreover, EP significantly increased MSC viability at higher concentrations [Figure 1(a)]. The final concentrations selected for all subsequent assays in our study were 15 µM and 50 µM [Figure 1(b)].

Figure 1.

Viability of MSCs after exposure to increasing concentrations of Eltrombopag over 24 h. The average absorbance values of control untreated samples were taken as 100%. Data are summarized as medians. (a) In vitro MTT assays of MSCs from HD (n = 7). (b) In vitro MTT assays with selected concentrations for subsequent experiments (n = 17).

Eltrombopag inhibits the adipogenic differentiation of MSCs

After appropriate expansion in the specific differentiation media, all MSC samples from both experimental groups were differentiated into either adipocytes or osteoblasts [Figure 2(a)].

Figure 2.

Differentiation ability of MSC treated with Eltrombopag. (a) Adipogenic and osteogenic differentiation, control and differentiated MSCs treated with 15 µM and 50 µM EP, untreated. 10X objective (n = 7). (b) Weekly number of adipocytes per field. Data are summarized as medians (n = 7). (c) RT-PCR genes involved in differentiation. MSCs treated with EP and untreated, differentiated for 21 days with specific media (n = 4).

As shown in Figure 2(b), when MSCs were treated with EP, their adipogenic differentiation capacity significantly decreased, this reduction being significantly greater with EP at 50 µM. There were no significant differences in the expression of the main genes involved in adipogenic and osteogenic differentiation.

Eltrombopag modifies the expression of genes involved in hypoxia, metabolism and other functions

The differential expression analysis of MSCs treated at 15 µM, 50 µM of EP and untreated MSC concentration is shown in Figure 3(a). Most of the differentiated genes did not vary as a function of EP concentration (FDR < 0.05) so the concentration at 15 µM was chosen for this analysis, in which 71 and 25 genes, respectively, were overexpressed and underexpressed relative to the control. Table 2 shows the top 10 most overexpressed genes, which were also found at 50 µM. The GEO accession number for the transcriptome data reported in this paper is GSE202127.

Figure 3.

Differential gene expression profiles of MSCs obtained after treatment with Eltrombopag (EP). (a) Heatmap showing the expression profiles of the genes that presented a significant change in MSCs treated with EP 15 µM, EP 50 µM or vehicle control (CTL, 0 µM) (n = 4 donors). The heatmap was constructed after supervised hierarchical clustering of expression profiles of the MSCs and includes 974 genes differentially expressed under the three conditions. A color bar indicates the normalized expression signal (from 1 to 20) over a log₂ scale, with blue and red indicating low and high levels of expression, respectively, and white representing values around the median. (b) Functional enrichment analysis was done using the list of the top 200 genes that were upregulated when MSCs were treated with 15 µM of EP. The bar plot includes the main upregulated functions ordered by their enrichment ratio. All these functions correspond to GO-BP terms and KEGG pathways; all of them were significant (FDR < 0.05). (c) Quantitative real-time polymerase chain reaction (qRT-PCR) in independent samples of MSCs isolated from bone marrow: without treatment (0 µM), or treated with 15 µM and 50 µM EP. Results are summarized as medians (7 samples tested).

Table 2.

Top-ten genes overexpressed in Eltrombopag-treated MSC (15 µM).

Gene symbol	ID	Fold change	p	FDR p
BNIP3L	TC0800007080.hg.1	4.67	4.08E-08	0.0003
KDM3A	TC0200008291.hg.1	4.89	2.24E-08	0.0003
BNIP3	TC1000012329.hg.1	7.56	3.47E-08	0.0003
TMEM45A	TC0300008148.hg.1	5.87	7.31E-08	0.0004
AK4	TC0100008618.hg.1	10.18	0.000000168	0.0005
GYS1	TC0800007080.hg.1	10.22	0.000000126	0.0005
PGK1	TC0X00007709.hg.1	7.91	0.000000149	0.0005
SLC2A1	TC0100013908.hg.1	9.23	0.000000184	0.0005
PDK1	TC0200009978.hg.1	9.42	0.000000242	0.0006
VEGFA	TC0600008109.hg.1	5.31	0.000000736	0.0016

Some of these genes are involved in the cellular response to hypoxia and negative regulation in apoptosis processes (VEGFA, BNIP3 L, BNIP3), in oxidation and reduction processes (KDM3A), in metabolism and antibiotic biosynthesis pathways (AK4, PGK1), in AMPK signaling pathways (GYS1), as well as in the HIF-1 signaling pathway (PDK1, VEGFA, SLC2A1) and in glycogenesis. Focusing on the top-two underexpressed genes, they were also involved in some of the previous pathways: apoptosis (BDH2) and HIF1α.

Functional enrichment analysis was done using the WebGestalt bioinformatic server (http://www.webgestalt.org/), searching two functional annotation databases (Gene Ontology GO-BP and KEGG pathways) with the list of the top 200 genes that were upregulated when MSCs were treated with EP. The top 30 biological-functional terms obtained in this enrichment were selected (all having an adjusted FDR value p < 0.05) [Figure 3(a)]. The most significant altered functions corresponded to glycolysis, glucose catabolism and the generation of ATP and NADH, suggesting a strong upregulation of cellular energy and respiration metabolism. Moreover, the presence of several biological terms indicating response to oxygen and response to hypoxia confirms that respiration and cellular oxygen homeostasis are affected in these cells by their treatment with EP [Figure 3(b)].

The expression of most of these genes was confirmed by RT-PCR [Figure 3(c)], as was as the underexpression of BDH2 involved in cellular cycle, differentiation, and apoptosis. HIF1α underexpression activates many genes associated with energy metabolism, angiogenesis, apoptosis and other genes involved in increasing oxygen release and facilitating adaptation to hypoxia.

Eltrombopag does not increase intercellular ROS level or affect DNA damage

Analysis showed that the drug did not increase the intracellular levels of ROS, although ROS levels were lower at higher doses [Figure 4(a)]. Likewise, the results of DNA double-strand breaks analyses do not support the presence of cell damage in MSCs treated with the EP [Figure 4(b)].

Figure 4.

Functional assays: (a) median fluorescence intensity of the intracytoplasmic ROS levels (n = 7), (b) percentage of cells with DNA damage (n = 5), and (c) absolute number of granulo-monocyte colonies (n = 11).

Eltrombopag increases in vitro hematopoietic supporting ability of MSCs

More granulo-monocytic colonies (CFU-GMs) were induced from healthy CD34⁺ cells that had been previously co-cultured with MSCs treated with EP than from those that had been co-cultured with MSCs in the absence of the drug [Figure 4(c)].

Discussion

The originality of the present work lies in it addressing, for the first time, the effects of EP on the hematopoietic supporting ability of healthy MSCs, which are key cells in the regulation of hematopoiesis. The role of EP in stimulating hematopoiesis in bone marrow failure has been clinically validated.^{1,4,5,11,25,26} Nevertheless, besides the well-known direct actions of this drug on hematopoietic progenitor cells, our study helps show how EP exerts some interesting effects on the stromal compartment that can also favor the hematopoietic process.²

We have shown here for the first time how EP can act on MSCs, increasing their proliferation and osteogenic differentiation capacity while decreasing their adipogenic differentiation ability. In addition, expression arrays revealed the overexpression of genes involved in pathways related to hypoxia, inhibition of apoptosis, energy metabolism and hematopoiesis. These were confirmed by RT-PCR. Functional assays also identified a decrease in oxidative stress in MSCs treated with high doses of the drug. Furthermore, the drug did not induce any significant DNA damage within the cell. Finally, clonogenic assays confirmed that MSCs treated with EP were better able to support hematopoiesis in vitro. Although some of these observations are based on small sample sizes, there were enough cases to enable significant statistical differences to be detected, and to validate the genetic profile study by RT-PCR. An interesting finding of our work is that EP at a concentration of 50 µM increases the viability of MSCs measured by the MTT assay, whereas at 15 µM the viability is similar to that of untreated MSCs. The selection of the concentrations to be used in our experiments requires further comment. Most analyses of the role of EP in megakaryopoiesis and hematopoiesis in vitro use concentrations ranging from 0.1 to 30 µg/ml in samples from healthy donors or patients.²⁷ In the current study, we initially tested concentrations from 10 to 50 µM. In subsequent studies, we decided to focus primarily on the 15-µM concentration, since it is widely used and is similar to the peak concentration of the drug in plasma of healthy subjects.²⁸ However, we also wanted to evaluate the effects of EP at 50 µM, since, as we have just mentioned, this concentration significantly increased the viability of MSCs. In a recent study, Di Paola et al.¹⁷ have assessed the effects of 6 µM of EP on MSCs of ITP pediatric patients, showing that the drug is able to increase viability and proliferation, while decreasing the release of IFN-gamma and IL-6 in MSCs from these subjects. The effects of the drug on healthy-donor-derived MSCs (also of pediatric age) were minimal or absent at the evaluated concentration.

The recent description that EP may exert its effects on MSCs from ITP patients by interfering with intracellular iron signaling is evidence of the existence of alternative mechanisms of action of this drug on different cell types.¹⁷ Kurokawa et al.²⁹ described that EP has the capacity to inhibit the proliferation of non-myeloid tumor cell lines through other mechanisms that do not involve the TPO receptor. Other authors have reported that EP does not increase the growth of bone marrow cells from patients with MDS/AML.²⁷ This selective finding about proliferation depending on the cell type is very intriguing and warrants further study.

When assessing one of the functional hallmarks of human MSCs, such as proliferation and multilineage differentiation, our studies revealed that EP increased MSC proliferation and differentiation with other bone marrow niche cells. The intimate interaction between HSCs and the BM microenvironment is essential for maintaining and regulating the behavior and properties of HSCs throughout an individual’s lifetime.^30–32 Among the minimal criteria for defining ISTC, MSCs should be capable of differentiating in vitro into adipocytes and osteoblasts.³³ In our study, MSCs treated with EP differentiated to osteocytes and adipocytes, although we observed an imbalance in the osteogenic/adipogenic differentiation ability compared with untreated cells. There were significantly fewer adipocytes in the EP-cultured cells. In addition, a tendency toward the stronger expression of genes involved in mineralization and osteogenesis, such as SPARC, SPP1 and RUNX2, as well as a decrease in the level of expression of AEBP1. It has been widely described that the number of adipocytes is inversely related to hematopoietic activity.³⁴ Autophagy or the liposomal degradation pathway regulates cellular homeostasis. This process has been linked to various age-associated diseases, including cancer. There is evidence that autophagy may also play an important role during cell differentiation and in the maintenance of hematopoietic stem progenitor cells (HSPCs) and in decreasing cellular stress.^35,36 Data confirmed overexpression of genes such as BNIP3 that may stimulate autophagy in the stroma and prevent MSCs aging. Its overexpression could be related to the reduction in ROS. Osteogenic differentiation in MSCs from bone marrow is favored by autophagy.³⁷ In addition, Lee and Joe³⁸ have demonstrated that hypoxic conditions can enhance the therapeutic efficacy of MSCs by increasing the level of secretion cytokines and autophagy.

Several genes overexpressed in our study, such as BNIP3, PGK1, TMEM45A and VEGFa, are known to be involved in response to hypoxia. Under hypoxic conditions, the maintenance of hematopoietic stem cells becomes stricter, so that the clonogenic capacity, proliferation and differentiation of these cells and MSCs increases.³⁹ Moreover, under these same conditions there is an increase in the level of expression of the VEGF-a in BM-MSCs.⁴⁰ Hematopoiesis and osteogenesis are both increased, and adipogenic differentiation is reduced, under hypoxic conditions.^2,41 Our results have shown that EP induces hypoxic conditions in MSCs, and that these facilitate the development of new therapeutic targets.

Chemically, EP has a central metal group that allows it to bind intracellular iron, thereby modifying iron homeostasis.⁴² This drug causes molecular changes in HSCs that are capable of restoring intracellular iron concentrations and reducing oxidative stress levels.⁴ Although further studies are needed to understand the effects of EP on reactive oxygen species, we have observed a drop in oxidative stress levels in treated MSCs. Iron plays an important role in hematopoiesis. However, its accumulation in certain diseases such as myelodysplastic syndromes or in patients undergoing transplantation increases the levels of intracellular ROS, thereby increasing oxidative stress and decreasing the hematopoietic capacity of the microenvironment and the degree of differentiation of the CD34⁺ HSCs.⁴³ Several authors^2,44 have studied ROS levels in malignant hematopoietic cells and revealed the depletion of these hematopoietic progenitors in patients. The use of EP in preclinical trials reduces the extent of intracellular ROS accumulation, intracellular free iron levels, and the level of oxidative stress in several cell types.^4,10,45 Hernández-Sánchez et al.⁴⁶ showed that ITP patients who did not respond to EP treatment presented several genes involved in activating the heme pathway. This could be related to an increase in the levels of intracellular iron, oxidative stress (ROS) and ineffective hematopoiesis. These effects were not present in drug-responsive patients. The latter findings are all consistent with our results, even though the cell type assessed is different.

Previous studies in MSCs have shown that excessive ROS can alter self-renewal, favor adipogenesis, and inhibit osteogenesis, proliferation and immunomodulatory properties.^47,48 Our results suggest that EP can reverse all these effects. In the process of differentiating hematopoietic progenitor cells toward megakaryocytic lineage, MSCs are involved and are able to secrete several factors, such as thrombopoietin, which is import for forming megakaryocytes.^12,49,50

In our study, EP did not produce DNA damage in MSC. EP inhibited DNA replication in cells from Ewing’s sarcoma.²⁶ However, several articles conclude that it would be worthwhile to explore further the drug’s ability to stimulate DNA damage repair in abnormal cells.

Finally, the role of MSCs in supporting hematopoiesis is essential for regulating this process.^9,51 Our observation that EP-treated MSCs can increase the clonogenic capacity of CD34⁺ HSCs is probably the most important finding of this study. It provides evidence in favor of using the drug in cases where hematopoiesis is defective, besides their well-known direct effects on hematopoietic cells, and implies that further studies on the effects of EP on MSC from patients with aplastic anemia are warranted.

Footnotes

Acknowledgements

The authors would like to thank Rebeca Ortega of the University Hospital of Salamanca for her technical assistance in the differentiation assays, and Phil Mason for his support with improving the English language of the manuscript.

Declarations

ORCID iDs

Sandra Muntión

Fermín Sánchez-Guijo

References

Bussel

Kulasekararaj

Cooper

, et al. Mechanisms and therapeutic prospects of thrombopoietin receptor agonists. Semin Hematol 2019; 56: 262–278.

Chen

Wang

Zhou

, et al. Advances in the understanding of poor graft function following allogeneic hematopoietic stem-cell transplantation. Ther Adv Hematol 2020; 11: 2040620720948743.

Kaushansky

. Molecular mechanisms of thrombopoietin signaling. J Thromb Haemost 2009; 7(Suppl. 1): 235–238.

Kao

Chen

Narayanagari

, et al. Thrombopoietin receptor-independent stimulation of hematopoietic stem cells by eltrombopag. Science Translational Medicine 2018; 10: eaas9563.

Sun

Tsai

Nowak

, et al. Eltrombopag, a thrombopoietin receptor agonist, enhances human umbilical cord blood hematopoietic stem/primitive progenitor cell expansion and promotes multi-lineage hematopoiesis. Stem Cell Res 2012; 9: 77–86.

Rivera

Bastida

Lopez-Corral

, et al. Usefulness of eltrombopag for treating thrombocytopenia after allogeneic stem cell transplantation. Bone Marrow Transplant 2019; 54: 757–761.

Gilreath

Bubalo

. Thrombopoietin receptor agonists (TPO-RAs): drug class considerations for pharmacists. Drugs 2021; 81: 1285–1305, https://link.springer.com/10.1007/s40265-021-01553-7

Desmond

Townsley

Dumitriu

, et al. Eltrombopag restores trilineage hematopoiesis in refractory severe aplastic anemia that can be sustained on discontinuation of drug. Blood 2014; 123: 1818–1825, https://ashpublications.org/blood/article/123/12/1818/32724/Eltrombopag-restores-trilineage-hematopoiesis-in

Dazzi

Ramasamy

Glennie

, et al. The role of mesenchymal stem cells in haemopoiesis. Blood Rev 2006; 20: 161–171.

10.

Pérez-Simón

Tabera

Sarasquete

, et al. Mesenchymal stem cells are functionally abnormal in patients with immune thrombocytopenic purpura. Cytotherapy 2009;11: 698–705.

11.

Zhang

, et al. The defective bone marrow-derived mesenchymal stem cells in patients with chronic immune thrombocytopenia. Autoimmunity 2014; 47: 519–529.

12.

Feng

, et al. Mesenchymal stem cell deficiency influences megakaryocytopoiesis through the TNFAIP3/NF-κB/SMAD pathway in patients with immune thrombocytopenia. Br J Haematol 2018; 180: 395–411.

13.

Guan

Sun

, et al. Role of bone marrow-derived mesenchymal stem cell defects in CD8+CD28– suppressor T-lymphocyte induction in patients with immune thrombocytopenia and associated mechanisms. Br J Haematol 2020; 191: 852–862.

14.

Huo

Zhang

Ren

, et al. Multifaceted characterization of the signatures and efficacy of mesenchymal stem/stromal cells in acquired aplastic anemia. Stem Cell Res Ther 2020; 11: 59.

15.

Yang

, et al. Differential gene expression profile associated with the abnormality of bone marrow mesenchymal stem cells in aplastic anemia. PLoS ONE 2012; 7: e47764.

16.

Sharma

Rawat

Gupta

, et al. Human acquired aplastic anemia patients’ bone-marrow-derived mesenchymal stem cells are not influenced by hematopoietic compartment and maintain stemness and immune properties. Anemia 2021; 2021: 6678067.

17.

Di Paola

Palumbo

Tortora

, et al. Eltrombopag in paediatric immune thrombocytopenia: Iron metabolism modulation in mesenchymal stromal cells. Br J Haematol 2022; 197: 110–119.

18.

MuntiÃ³n

Ramos

Diez-Campelo

, et al. Microvesicles from mesenchymal stromal cells are involved in hpc-microenvironment crosstalk in myelodysplastic patients. PLoS ONE 2016; 11: e0146722.

19.

Paíno

Sarasquete

Paiva

, et al. Phenotypic, genomic and functional characterization reveals no differences between CD138++ and CD138low subpopulations in multiple myeloma cell lines. PLoS ONE 2014; 9: e92378.

20.

Carrancio

López-Holgado

Sánchez-Guijo

, et al. Optimization of mesenchymal stem cell expansion procedures by cell separation and culture conditions modification. Exp Hematol 2008; 36: 1014–1021.

21.

Gregory

Gunn

Peister

, et al. An Alizarin red-based assay of mineralization by adherent cells in culture: comparison with cetylpyridinium chloride extraction. Anal Biochem 2004; 329: 77–84.

22.

Stelzer

Rosen

Plaschkes

, et al. The GeneCards suite: From gene data mining to disease genome sequence analyses. Curr Protoc Bioinformatics 2016; 54: 1.30.1–1.30.33.

23.

Huang da

Sherman

Lempicki

. Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat Protoc 2009; 4: 44–57.

24.

Preciado

Sirerol-Piquer

Muntión

, et al. Co-administration of human MSC overexpressing HIF-1α increases human CD34+ cell engraftment in vivo. Stem Cell Res Ther 2021; 12: 601.

25.

Jeong

Levine

Abayasekara

, et al. The non-peptide thrombopoietin receptor agonist eltrombopag stimulates megakaryopoiesis in bone marrow cells from patients with relapsed multiple myeloma. J Hematol Oncol 2015; 8: 37.

26.

Waters

Goss

Koppenhafer

, et al. Eltrombopag inhibits the proliferation of Ewing sarcoma cells via iron chelation and impaired DNA replication. BMC Cancer 2020; 20: 1171.

27.

Will

Kawahara

Luciano

, et al. Effect of the nonpeptide thrombopoietin receptor agonist eltrombopag on bone marrow cells from patients with acute myeloid leukemia and myelodysplastic syndrome. Blood 2009; 114: 3899–3908.

28.

Erhardt

Erickson-Miller

Aivado

, et al. Comparative analyses of the small molecule thrombopoietin receptor agonist eltrombopag and thrombopoietin on in vitro platelet function. Exp Hematol 2009; 37: 1030–1037.

29.

Kurokawa

Murata

Zheng

, et al. The eltrombopag antitumor effect on hepatocellular carcinoma. Int J Oncol 2015; 47: 1696–1702.

30.

Boulais

Frenette

. Making sense of hematopoietic stem cell niches. Blood 2015; 125: 2621–2629, http://ashpublications.org/blood/article-pdf/125/17/2621/1384676/2621.pdf

31.

Kunisaki

. The hematopoietic stem cell niche in homeostasis and disease. Rinsho- ketsueki 2015; 56: 1888–1893, http://ashpublications.org/blood/article-pdf/126/22/2443/1390420/2443.pdf

32.

Pinho

Frenette

. Haematopoietic stem cell activity and interactions with the niche. Nat Rev Mol Cell Biol 2019; 20: 303–320, http://www.nature.com/articles/s41580-019-0103-9

33.

Dominici

Le Blanc

Mueller

, et al. Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy Position Statement. Cytotherapy 2006; 8: 315–317, https://pubmed.ncbi.nlm.nih.gov/16923606/(accessed 13 September 2021).

34.

Naveiras

Nardi

Wenzel

, et al. Bone-marrow adipocytes as negative regulators of the haematopoietic microenvironment. Nature 2009; 460: 259–263, http://www.nature.com/articles/nature08099

35.

Gomez-Puerto

Folkerts

Wierenga

, et al. Autophagy proteins atg5 and atg7 are essential for the maintenance of human cd34+ hematopoietic stem-progenitor cells. Stem Cells 2016; 34: 1651–1663, https://academic.oup.com/stmcls/article/34/6/1651-1663/6408274

36.

Zhang

Ney

. Role of BNIP3 and NIX in cell death, autophagy, and mitophagy. Cell Death Differ 2009; 16: 939–946.

37.

Wan

Zhuo

, et al. Autophagy promotes osteogenic differentiation of human bone marrow mesenchymal stem cell derived from osteoporotic vertebrae. Biochem Biophys Res Commun 2017; 488(1): 46–52, https://linkinghub.elsevier.com/retrieve/pii/S0166432817301699

38.

Lee

Joe

. Autophagy mediates enhancement of proangiogenic activity by hypoxia in mesenchymal stromal/stem cells. Biochem Biophys Res Commun 2018; 501: 941–947.

39.

Martin-Rendon

Hale

Ryan

, et al. Transcriptional Profiling of human cord blood cd133+ and cultured bone marrow mesenchymal stem cells in response to hypoxia. Stem Cells 2007; 25: 1003–1012.

40.

Liu

Hao

Xia

, et al. Hypoxia pretreatment of bone marrow mesenchymal stem cells facilitates angiogenesis by improving the function of endothelial cells in diabetic rats with lower ischemia. PLoS ONE 2015; 10: e0126715, https://dx.plos.org/10.1371/journal.pone.0126715

41.

Wagegg

Gaber

Lohanatha

, et al. Hypoxia promotes osteogenesis but suppresses adipogenesis of human mesenchymal stromal cells in a hypoxia-inducible factor-1 dependent manner. PLoS ONE 2012; 7: e46483.

42.

Kuter

. Biology and Chemistry of Thrombopoietic Agents. Semin Hematol 2010; 47: 243–248, https://linkinghub.elsevier.com/retrieve/pii/S0037196310000302

43.

Isidori

Borin

Elli

, et al. Iron toxicity – Its effect on the bone marrow. Blood Rev 2018; 32: 473–479.

44.

Jiménez-Solas

López-Cadenas

Aires-Mejía

, et al. Deferasirox reduces oxidative DNA damage in bone marrow cells from myelodysplastic patients and improves their differentiation capacity. Br J Haematol 2019; 187: 93–104.

45.

Ono-Uruga

Ikeda

Matsubara

. Platelet production using adipose-derived mesenchymal stem cells: mechanistic studies and clinical application. J Thromb Haemost 2021; 19: 342–350.

46.

Hernández-Sánchez

Bastida

Alonso-López

, et al. Transcriptomic analysis of patients with immune thrombocytopenia treated with eltrombopag. Platelets 2020; 31: 993–1000.

47.

Atashi

Modarressi

Pepper

. The role of reactive oxygen species in mesenchymal stem cell adipogenic and osteogenic differentiation: a review. Stem Cells Dev 2015; 24: 1150–1163.

48.

Denu

Hematti

. Effects of oxidative stress on mesenchymal stem cell biology. Oxid Med Cell Longev 2016; 2016: 2989076.

49.

Tozawa

Ono-Uruga

Yazawa

, et al. Megakaryocytes and platelets from a novel human adipose tissue-derived mesenchymal stem cell line. Blood 2019; 133: 633–643, http://ashpublications.org/blood/article-pdf/133/7/633/1552504/blood842641.pdf

50.

Zuo

Sun

Wang

, et al. Establishment of a novel mesenchymal stem cell-based regimen for chronic myeloid leukemia differentiation therapy. Cell Death Dis 2021; 12: 208.

51.

Méndez-Ferrer

Michurina

Ferraro

, et al. Mesenchymal and haematopoietic stem cells form a unique bone marrow niche. Nature 2010; 466: 829–834.

Eltrombopag increases the hematopoietic supporting ability of mesenchymal stem/stromal cells

Abstract

Background:

Objectives:

Design/Methods:

Results:

Conclusion:

Keywords

Introduction

Methods

Reagents

Patient samples

Cell viability assays

MSC differentiation ability

Adipocytic differentiation

Osteogenic differentiation

Mineralized bone quantification

Reverse transcription real-time PCR analyses of genes involved in MSC differentiation

Gene expression array

Gene expression analysis by RT-PCR

Functional assays

Intracellular reactive oxygen species (ROS) levels

DNA damage analysis

Clonogenic capacity

Statistical tests

Results

Eltrombopag increases the viability of MSCs

Eltrombopag inhibits the adipogenic differentiation of MSCs

Eltrombopag modifies the expression of genes involved in hypoxia, metabolism and other functions

Eltrombopag does not increase intercellular ROS level or affect DNA damage

Eltrombopag increases in vitro hematopoietic supporting ability of MSCs

Discussion

Footnotes

Acknowledgements

Declarations

ORCID iDs

References