Lusutrombopag or hetrombopag supports in vitro megakaryopoiesis better than other thrombopoietin receptor agonists

Thrombopoietin receptor agonists (TPORAs) have been used to treat thrombocytopenia patients with idiopathic thrombocytopenic purpura, aplastic anaemia and chronic liver disease and also after haematopoietic stem cell transplantation.1 There are many TPORAs such as eltrombopag (EPAG),2 avatrombopag (APAG),3 lusutrombopag (LPAG)4 and hetrombopag (HPAG).5 It is difficult to compare their effects in healthy volunteers and patients. In this study, we compared the in vitro effects of EPAG, APAG, LPAG and HPAG on human megakaryocyte (Mk) generation from mononuclear cells (MNCs) of umbilical cord blood (CB) in our originally developed serum-free condition. Here, we report that LPAG and HPAG were similarly effective and significantly more effective than EPAG, APAG, romiplostim or even thrombopoietin in support of megakaryopoiesis. Taken into account these observations, clinicians may choose one of the TPORAs for the treatment of patients. Culture conditions for human Mks and their progenitors are poorly defined. We thus optimized our chemically defined serum-free culture using CB which was provided by Tianjin Cord Blood Stem Cell Bank (Tianjin, China) after written informed consents were signed by parents. All procedures of this study were approved by the Institutional Ethics Committee of the Institute of Hematology and Blood Diseases Hospital. Our serum-free medium was originally developed for mouse HSC culture6, 7 and slightly modified for human Mk culture as follows: 0.5 mg/mL recombinant human albumin (rHSA, Albumin Bioscience), 1× insulin-transferrin-selenium-ethanolamine (Thermo Fisher Scientific), 5 × 10−5 M 2-mercaptoethanol (Thermo Fisher Scientific), 2 mM L-glutamine (Thermo Fisher Scientific), 0.1 mM minimum essential medium non-essential amino acids (Thermo Fisher Scientific), 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (Sigma Aldrich), 0.5 mg/mL penicillin-streptomycin-glutamine (Thermo Fisher Scientific) and 1 μM hydrocortisone (Sigma Aldrich) in Iscove's Modified Dulbecco's Medium (Gibco). Red blood cells were deleted from CB by 6% hydroxyethyl starch (HESPAN, B. Braun Medical). MNCs separated by sample density separator (TBD Science) were incubated in the serum-free condition at 37°C with 5% CO2 in the air. To detect Mks in culture, cells were stained with Alexa Fluor 700-anti-CD41, peridinin-chlorophyll-protein/cyanine 5.5-CD42b (HIP1, BioLegend), phycoerythrin/cyanine 7-anti-CD14 (63D3, BioLegend), Brilliant Violet 650-anti-CD16 (3G8, BioLegend) and phycoerythrin-anti-CD235a (HIR2, BD Biosciences) antibodies. Cells were resuspended in phosphate-buffered saline with 4′,6-diamidino-2-phenylindole (DAPI) with the appropriate number of Trucount Absolute Counting Beads (BD Biosciences) and analysed by FACSAriaIII (BD Biosciences). Figure S1 shows an example of our serum-free Mk culture system. CB MNCs were cultured with LPAG (MedChemExpress). CD41+CD42b+ cells in culture were detected by flow cytometry. The number of CD41+CD42b+ cells increased until days 12–15 of culture (Figure S1A). The majority of cultured cells appeared to be relatively small with 2 N (Figure S1A,B). To test the function of cultured cells, day 12 cultured cells were transplanted into NOD.Cg-PrkdcscidIL2rgtm1Sug/JicCrl (NOG) mice (Beijing Vital River Laboratory Animal Technology, China) irradiated at a dose of 2.0 Gy. A low but substantial level of human platelet reconstitution was detected (Figure S1C). Based on these results, we defined CD41+CD42b− cells and CD41+CD42b+ cells in culture as immature and mature Mks, respectively. Polyvinyl alcohol (PVA)8 and Soluplus9 have been used as serum substitutes for serum-free culture of human HSCs. We compared PVA (JAPAN VAM & POVAL, Osaka, Japan) and Soluplus (kindly provided by BASF, Ludwigshafen, Germany) with rHSA in human megakaryopoiesis in vitro (Figure S2). The total number of cells in rHSA culture was significantly greater than that in Soluplus culture which was significantly greater than that in PVA culture. rHSA supported the generation of CD41+CD42b+ mature Mks significantly better than did PVA or Soluplus. We concluded that rHSA can support Mk generation significantly better than does Soluplus or PVA in serum-free culture, consistent with our recent study reporting that rHSA supports mouse megakaryopoiesis better than PVA.10 We compared the effect of LPAG with that of thrombopoietin (TPO) or romiplostim (Figure S3). Interestingly, LPAG supported in vitro generation of immature and mature Mks from CB significantly better than TPO or romiplostim. We then compared the effects of EPAG (MedChemExpress) and HPAG (kindly provided by Hengrui, China) and those of APAG (MedChemExpress) and LPAG at different concentrations in rHSA-based medium. The numbers of immature and mature Mk increased in a dose-dependent manner except for LPAG, which showed the largest number at 0.2 μM. HPAG at 0.1 μg/mL supported Mk generation significantly greater than EPAG at 0.5 μg/mL (Figure 1A–C). LPAG at 0.2 μM supported Mk generation significantly greater than APAG at 1.0 μM (Figure 1D–F). Higher concentrations of all these four TPORAs seemed to have a negative effect in Mk culture. To confirm the optimal concentrations, CD34+ cells were sorted from CB MNCs after stained with APC-anti-CD34 antibody (BD Biosciences) by FACSAriaIII and cultured at different concentrations of LPAG or HPAG (Figure S4). Consistent with the culture of MNCs, the optimal concentration of LPAG appeared to be 0.2–1.0 μM. The optimal concentration of HPAG appeared to be 0.05–0.1 μg/mL. We then directly compared the effects of 0.2 μM LPAG and 0.05 μg/mL HPAG, using CD34+CD110lowCD117low cells. Their effects on Mk generation were similar (Figure S5). Taken together, we concluded that either LPAG or HPAG supports megakaryopoiesis more efficiently than either EPAG or APAG. LPAG or HPAG would be a better choice for severe cases of aplastic anaemia. To characterize cultured Mks, we performed single-cell RNA sequencing. CB MNCs were cultured with HPAG for 12 days. Single-cell RNA-seq libraries were constructed with Chromium Next GEM Single Cell 3'Reagent Kits v3.1 (10X Genomics, Pleasanton, CA), according to the manufacturer's instructions,11 and sequenced on an Illumina Hiseq X Ten platform at Novogene (Tianjin, China). Sequencing raw data were quantified by Cell Ranger (version 7.1) software with default settings and aligned to the human reference genome GRCh38. The Seurat package (version 4.2.1) was used for mapping analysis, cell clustering, dimensional reduction and differentially expressed gene analysis. Both total live cells and CD41+CD42b+ cells were simultaneously analysed (Figure 2). These cells showed similar clustering by uniform manifold approximation and projection (UMAP) analysis12 (Figure 2A), although the proportions of Clusters 0, 1, 2 and 3 were larger in CD41+CD42b+ cells (Figure 2B). Mk maker genes were similarly distributed in the clusters between live cells and CD41+CD42b+ cells (Figure S6). The clusters in this study overlapped hBM-MK1-4 which have been previously identified as Mk clusters in human bone marrow (Figure 2C),13 suggesting that heterogeneous Mks were generated in culture. Cultured Mks seemed to be not so mature in terms of morphology and polymerization, but transcriptionally similar to Mk in bone marrow. Unlike TPO which binds the extracellular domain of the human TPO receptor, species-specific TPORAs commonly bind the transmembrane region of the receptor4, 14 and initiate intracellular signalling by receptor dimerization at 499 Histone.15 LPAG, HPAG, EPAG and APAG all supported in vitro megakaryopoiesis, but at different degrees. Binding affinity may differ among these ligands. Further studies of signal transduction are required to clarify their different actions. Nevertheless, their efficacies differed in terms of in vitro production of Mks, providing useful information for their clinical use. Yunfang He, Jing Hu, Jinglei Zhai, Lisha Wang, Xuelian Cheng and Jinhong Wang, performed experiments; Fang Dong, Xin Zhao, Fengkui Zhang and Hideo Ema supervised the study; and Yunfang He, Jing Hu, Fengkui Zhang and Hideo Ema wrote the manuscript. We thank Norio Komatsu for critical reading of the manuscript. This study was supported by the National Key R&D Program of China (2022YFA110330, 2022YFA1103500), the National Natural Science Foundation of China (82170118, 82370116, 82222004), the Haihe Laboratory of Cell Ecosystem Innovation Fund (22HHXBSS00038), Chinese Academy of Medical Sciences (CAMS) Innovation Fund for Medical Sciences (2021-I2M-1-073, 2024-I2M-TS-024), Distinguished Young Scholars of Tianjin (23JCJQJC00220) and the Science, Technology & Innovation Project of the Xiongan New Area (2022XAGG0142). The authors have no conflicts of interest to report. The National Natural Science Foundation of China, Chinese Academy of Medical Sciences, Haihe Laboratory of Cell Ecosystem, Chinese Academy of Medical Sciences (CAMS) Innovation Fund for Medical Sciences, Distinguished Young Scholars of Tianjin and the Science, Technology & Innovation Project of the Xiongan New Area. Cord blood was provided by Tianjin Cord Blood Stem Cell Bank (Tianjin, China) after written informed consents were signed by parents. All procedures of this study were approved by the Institutional Ethics Committee of the Institute of Hematology and Blood Diseases Hospital. Single-cell RNA-seq data of cultured human megakaryocytes were deposited in Genome Sequence Archive (GAS-Human: HRA007883). The other data are available from the corresponding authors upon reasonable request. Figure S1. Figure S2. Figure S3. Figure S4. Figure S5. Figure S6. 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