The combination of machine learning and transcriptomics reveals a novel megakaryopoiesis inducer, MO-A, that promotes thrombopoiesis by activating FGF1/FGFR1/PI3K/Akt/NF-κB signaling.

Radiation-induced thrombocytopenia (RIT) occurs widely and causes high mortality and morbidity in cancer patients who receive radiotherapy. However, specific drugs for treating RIT remain woefully inadequate. Here, we first developed a drug screening model using naive Bayes, a machine learning (ML) algorithm, to virtually screen the active compounds promoting megakaryopoiesis and thrombopoiesis. A natural product library was screened by the model, and methylophiopogonanone A (MO-A) was identified as the most active compound. The activity of MO-A was then validated in vitro and showed that MO-A could markedly induce megakaryocyte (MK) differentiation of K562 and Meg-01 cells in a concentration-dependent manner. Furthermore, the therapeutic action of MO-A on RIT was evaluated, and MO-A significantly accelerated platelet level recovery, platelet activation, megakaryopoiesis, MK differentiation in RIT mice. Moreover, RNA-sequencing (RNA-seq) indicated that the PI3K cascade was closely related to MK differentiation induced by MO-A. Finally, experimental verification demonstrated that MO-A obviously induced the expression of FGF1 and FGFR1, and increased the phosphorylation of PI3K, Akt and NF-κB. Blocking FGFR1 with its inhibitor dovitinib suppressed MO-A-induced MK differentiation, and PI3K, Akt and NF-κB phosphorylation. Similarly, inhibition of PI3K-Akt signal pathway by its inhibitor LY294002 suppressed MK differentiation, and PI3K, Akt and NF-κB phosphorylation induced by MO-A. Taken together, our study provides an efficient drug discovery strategy for hematological diseases, and demonstrates that MO-A is a novel countermeasure for treating RIT through activation of the FGF1/FGFR1/PI3K/Akt/NF-κB signaling pathway.

Toll-like Receptors and Thrombopoiesis.

Platelets are the second most abundant blood component after red blood cells and can participate in a variety of physiological and pathological functions. Beyond its traditional role in hemostasis and thrombosis, it also plays an indispensable role in inflammatory diseases. However, thrombocytopenia is a common hematologic problem in the clinic, and it presents a proportional relationship with the fatality of many diseases. Therefore, the prevention and treatment of thrombocytopenia is of great importance. The expression of Toll-like receptors (TLRs) is one of the most relevant characteristics of thrombopoiesis and the platelet inflammatory function. We know that the TLR family is found on the surface or inside almost all cells, where they perform many immune functions. Of those, TLR2 and TLR4 are the main stress-inducing members and play an integral role in inflammatory diseases and platelet production and function. Therefore, the aim of this review is to present and discuss the relationship between platelets, inflammation and the TLR family and extend recent research on the influence of the TLR2 and TLR4 pathways and the regulation of platelet production and function. Reviewing the interaction between TLRs and platelets in inflammation may be a research direction or program for the treatment of thrombocytopenia-related and inflammatory-related diseases.

Novel chemical-structure TPOR agonist, TMEA, promotes megakaryocytes differentiation and thrombopoiesis via mTOR and ERK signalings.

BACKGROUND: Non-peptide thrombopoietin receptor (TPOR) agonists are promising therapies for the mitigation and treatment of thrombocytopenia. However, only few agents are available as safe and effective for stimulating platelet production for thrombocytopenic patients in the clinic. PURPOSE: This study aimed to develop a novel small molecule TPOR agonist and investigate its underlying regulation of function in megakaryocytes (MKs) differentiation and thrombopoiesis. METHODS: A potential active compound that promotes MKs differentiation and thrombopoiesis was obtained by machine learning (ML). Meanwhile, the effect was verified in zebrafish model, HEL and Meg-01 cells. Next, the key regulatory target was identified by Drug Affinity Responsive Target Stabilization Assay (DARTS), Cellular Thermal Shift Assay (CETSA), and molecular simulation experiments. After that, RNA-sequencing (RNA-seq) was used to further confirm the associated pathways and evaluate the gene expression induced during MK differentiation. In vivo, irradiation (IR) mice, C57BL/6N-TPORem1cyagen (Tpor-/-) mice were constructed by CRISPR/Cas9 technology to examine the therapeutic effect of TMEA on thrombocytopenia. RESULTS: A natural chemical-structure small molecule TMEA was predicted to be a potential active compound based on ML. Obvious phenotypes of MKs differentiation were observed by TMEA induction in zebrafish model and TMEA could increase co-expression of CD41/CD42b, DNA content, and promote polyploidization and maturation of MKs in HEL and Meg-01 cells. Mechanically, TMEA could bind with TPOR protein and further regulate the PI3K/AKT/mTOR/P70S6K and MEK/ERK signal pathways. In vivo, TMEA evidently promoted platelet regeneration in mice with radiation-induced thrombocytopenia but had no effect on Tpor-/- and C57BL/6 (WT) mice. CONCLUSION: TMEA could serve as a novel TPOR agonist to promote MKs differentiation and thrombopoiesis via mTOR and ERK signaling and could potentially be created as a promising new drug to treat thrombocytopenia.

Proteomic landscapes of inherited platelet disorders with different etiologies.

BACKGROUND: Inherited platelet disorders (IPDs) are a heterogeneous group of rare diseases that are caused by the defects in early megakaryopoiesis, proplatelet formation, and/or mature platelet function. Although genomic sequencing is increasingly used to identify genetic variants underlying IPD, this technique does not disclose resulting molecular changes that impact platelet function. Proteins are the functional units that shape platelet function; however, insights into how variants that cause IPDs impact platelet proteomes are limited. OBJECTIVES: The objective of this study was to profile the platelet proteomics signatures of IPDs. METHODS: We performed unbiased label-free quantitative mass spectrometry (MS)-based proteome profiling on platelets of 34 patients with IPDs with variants in 13 ISTH TIER1 genes that affect different stages of platelet development. RESULTS: In line with the phenotypical heterogeneity between IPDs, proteomes were diverse between IPDs. We observed extensive proteomic alterations in patients with a GFI1B variant and for genetic variants in genes encoding proteins that impact cytoskeletal processes (MYH9, TUBB1, and WAS). Using the diversity between IPDs, we clustered protein dynamics, revealing disrupted protein-protein complexes. This analysis furthermore grouped proteins with similar cellular function and location, classifying mitochondrial protein constituents and identifying both known and putative novel alpha granule associated proteins. CONCLUSIONS: With this study, we demonstrate a MS-based proteomics perspective to IPDs. By integrating the effects of IPDs that impact different aspects of platelet function, we dissected the biological contexts of protein alterations to gain further insights into the biology of platelet (dys)function.

Akt-mediated mitochondrial metabolism regulates proplatelet formation and platelet shedding post vasopressin exposure.

BACKGROUND: Platelet shedding from mature megakaryocytes (MKs) in thrombopoiesis is the critical step for elevating circulating platelets fast and efficiently, however, the underlying mechanism is still not well-illustrated, and the therapeutic targets and candidates are even less. OBJECTIVES: In order to investigate the mechanisms for platelet shedding after vasopressin treatment and find new therapeutic targets for thrombocytopenia. METHODS: Platelet production was evaluated both in vivo and in vitro after arginine vasopressin (AVP) administration. The underlying biological mechanism of AVP-triggered thrombopoiesis were then investigated by a series of molecular and bioinformatics techniques. RESULTS: it is observed that proplatelet formation and platelet shedding in the final stages of thrombopoiesis promoted by AVP, an endogenous hormone, can quickly increases peripheral platelets. This rapid elevation is thus able to speed up platelet recovery after radiation as expected. The mechanism analysis reveal that proplatelet formation and platelet release from mature MKs facilitated by AVP is mainly mediated by Akt-regulated mitochondrial metabolism. In particular, phosphorylated Akt regulates mitochondrial metabolism through driving the association of hexokinase-2 with mitochondrial voltage dependent anion channel-1 in AVP-mediated thrombopoiesis. Further studies suggest that this interaction is stabilized by IκBα, the expression of which is controlled by insulin-regulated membrane aminopeptidase. CONCLUSION: these data demonstrate that phosphorylated Akt-mediated mitochondrial metabolism regulates platelet shedding from MKs in response to AVP, which will provide new therapeutic targets and further drug discovery clues for thrombocytopenia treatment.

Roles of non-coding RNA in megakaryocytopoiesis and thrombopoiesis: new target therapies in ITP.

Noncoding RNAs (ncRNAs) are a group of RNA molecules that cannot encode proteins, and a better understanding of the complex interaction networks coordinated by ncRNAs will provide a theoretical basis for the development of therapeutics targeting the regulatory effects of ncRNAs. Platelets are produced upon the differentiation of hematopoietic stem cells into megakaryocytes, 1011 per day, and are renewed every 8-9 days. The process of thrombopoiesis is affected by multiple factors, in which ncRNAs also exert a significant regulatory role. This article reviewed the regulatory roles of ncRNAs, mainly microRNAs (miRNAs), circRNAs (circular RNAs), and long non-coding RNAs (lncRNAs), in thrombopoiesis in recent years as well as their roles in primary immune thrombocytopenia (ITP).

What is the context? Platelets are produced from progenitor cells named megakaryocytes (MKs) differentiated from bone marrow-derived hematopoietic stem cells (HSCs).Thrombopoiesis refers to the process by which platelet-producing MKs release platelet granules into peripheral blood under the shear force of blood flow for further development and maturation.The process of megakaryocytopoiesis and thrombopoiesis is affected by multiple factors, wherein some ncRNAs also exert a significant regulatory role.miRNAs/lncRNAs play a promising role in t primary immune thrombocytopenia (ITP).What is new? This article reviewed the regulatory roles of ncRNAs, mainly microRNAs (miRNAs), circRNAs (circular RNAs), and long non-coding RNAs (lncRNAs), in thrombopoiesis.This article also reviewed the roles of ncRNAs in ITP.What is the impact?Changes in ncRNA expression are associated with changes in the production of MKs, thrombopoiesis, and platelet function, which allows a new understanding of the pathogenesis of many congenital or acquired platelet-related diseases.

Don't you forget about me(gakaryocytes).

Platelets (small, anucleate cell fragments) derive from large precursor cells, megakaryocytes (MKs), that reside in the bone marrow. MKs emerge from hematopoietic stem cells in a complex differentiation process that involves cytoplasmic maturation, including the formation of the demarcation membrane system, and polyploidization. The main function of MKs is the generation of platelets, which predominantly occurs through the release of long, microtubule-rich proplatelets into vessel sinusoids. However, the idea of a 1-dimensional role of MKs as platelet precursors is currently being questioned because of advances in high-resolution microscopy and single-cell omics. On the one hand, recent findings suggest that proplatelet formation from bone marrow-derived MKs is not the only mechanism of platelet production, but that it may also occur through budding of the plasma membrane and in distant organs such as lung or liver. On the other hand, novel evidence suggests that MKs not only maintain physiological platelet levels but further contribute to bone marrow homeostasis through the release of extracellular vesicles or cytokines, such as transforming growth factor ß1 or platelet factor 4. The notion of multitasking MKs was reinforced in recent studies by using single-cell RNA sequencing approaches on MKs derived from adult and fetal bone marrow and lungs, leading to the identification of different MK subsets that appeared to exhibit immunomodulatory or secretory roles. In the following article, novel insights into the mechanisms leading to proplatelet formation in vitro and in vivo will be reviewed and the hypothesis of MKs as immunoregulatory cells will be critically discussed.

You reap what you sow: Neutrophils "plucking" platelets harvest prothrombotic effects.

Inflammatory insults affect platelet production, but it is yet unknown what mechanisms can drive rapid adaptations in thrombopoiesis. In this issue of Immunity, Petzold et al. (2022) propose that neutrophils "pluck" on megakaryocytes in the bone marrow to tune platelet release.

Targeting TLR2/Rac1/cdc42/JNK Pathway to Reveal That Ruxolitinib Promotes Thrombocytopoiesis.

BACKGROUND: Thrombocytopenia has long been considered an important complication of chemotherapy and radiotherapy, which severely limits the effectiveness of cancer treatment and the overall survival of patients. However, clinical treatment options are extremely limited so far. Ruxolitinib is a potential candidate. METHODS: The impact of ruxolitinib on the differentiation and maturation of K562 and Meg-01 cells megakaryocytes (MKs) was examined by flow cytometry, Giemsa and Phalloidin staining. A mouse model of radiation-injured thrombocytopenia (RIT) was employed to evaluate the action of ruxolitinib on thrombocytopoiesis. Network pharmacology, molecular docking, drug affinity responsive target stability assay (DARTS), RNA sequencing, protein blotting and immunofluorescence analysis were applied to explore the targets and mechanisms of action of ruxolitinib. RESULTS: Ruxolitinib can stimulate MK differentiation and maturation in a dose-dependent manner and accelerates recovery of MKs and thrombocytopoiesis in RIT mice. Biological targeting analysis showed that ruxolitinib binds directly to Toll Like Receptor 2 (TLR2) to activate Rac1/cdc42/JNK, and this action was shown to be blocked by C29, a specific inhibitor of TLR2. CONCLUSIONS: Ruxolitinib was first identified to facilitate MK differentiation and thrombocytopoiesis, which may alleviate RIT. The potential mechanism of ruxolitinib was to promote MK differentiation via activating the Rac1/cdc42/JNK pathway through binding to TLR2.

A New Role of NAP1L1 in Megakaryocytes and Human Platelets.

Platelets (PLTs) are anucleate and considered incapable of nuclear functions. Contrastingly, nuclear proteins were detected in human PLTs. For most of these proteins, it is unclear if nuclear or alternatively assigned functions are performed, a question we wanted to address for nuclear assembly protein 1like 1 (NAP1L1). Using a wide array of molecular methods, including RNAseq, co-IP, overexpression and functional assays, we explored expression pattern and functionality of NAP1L1 in PLTs, and CD34+-derived megakaryocytes (MKs). NAP1L1 is expressed in PLTs and MKs. Co-IP experiments revealed that dihydrolipolylysine-residue acetyltransferase (DLAT encoded protein PDC-E2, ODP2) dynamically interacts with NAP1L1. PDC-E2 is part of the mitochondrial pyruvate-dehydrogenase (PDH) multi-enzyme complex, playing a crucial role in maintaining cellular respiration, and promoting ATP-synthesis via the respiratory chain. Since altered mitochondrial function is a hallmark of infectious syndromes, we analyzed PDH activity in PLTs from septic patients demonstrating increased activity, paralleling NAP1L1 expression levels. MKs PDH activity decreased following an LPS-challenge. Furthermore, overexpression of NAP1L1 significantly altered the ability of MKs to form proplatelet extensions, diminishing thrombopoiesis. These results indicate that NAP1L1 performs in other than nucleosome-assembly functions in PTLs and MKs, binding a key mitochondrial protein as a potential chaperone, and gatekeeper, influencing PDH activity and thrombopoiesis.

Inflammatory platelet production stimulated by tyrosyl-tRNA synthetase mimicking viral infection.

Platelets play a role not only in hemostasis and thrombosis, but also in inflammation and innate immunity. We previously reported that an activated form of tyrosyl-tRNA synthetase (YRSACT) has an extratranslational activity that enhances megakaryopoiesis and platelet production in mice. Here, we report that YRSACT mimics inflammatory stress inducing a unique megakaryocyte (MK) population with stem cell (Sca1) and myeloid (F4/80) markers through a mechanism dependent on Toll-like receptor (TLR) activation and type I interferon (IFN-I) signaling. This mimicry of inflammatory stress by YRSACT was studied in mice infected by lymphocytic choriomeningitis virus (LCMV). Using Sca1/EGFP transgenic mice, we demonstrated that IFN-I induced by YRSACT or LCMV infection suppressed normal hematopoiesis while activating an alternative pathway of thrombopoiesis. Platelets of inflammatory origin (Sca1/EGFP+) were a relevant proportion of those circulating during recovery from thrombocytopenia. Analysis of these "inflammatory" MKs and platelets suggested their origin in myeloid/MK-biased hematopoietic stem cells (HSCs) that bypassed the classical MK-erythroid progenitor (MEP) pathway to replenish platelets and promote recovery from thrombocytopenia. Notably, inflammatory platelets displayed enhanced agonist-induced activation and procoagulant activities. Moreover, myeloid/MK-biased progenitors and MKs were mobilized from the bone marrow, as evidenced by their presence in the lung microvasculature within fibrin-containing microthrombi. Our results define the function of YRSACT in platelet generation and contribute to elucidate platelet alterations in number and function during viral infection.

A Novel Antithrombocytopenia Agent, Rhizoma cibotii, Promotes Megakaryopoiesis and Thrombopoiesis through the PI3K/AKT, MEK/ERK, and JAK2/STAT3 Signaling Pathways.

BACKGROUND: Cibotii rhizoma (CR) is a famous traditional Chinese medicine (TCM) used to treat bleeding, rheumatism, lumbago, etc. However, its therapeutic effects and mechanism against thrombocytopenia are still unknown so far. In the study, we investigated the effects of aqueous extracts of Cibotii rhizoma (AECRs) against thrombocytopenia and its molecular mechanism. METHODS: Giemsa staining, phalloidin staining, and flow cytometry were performed to measure the effect of AECRs on the megakaryocyte differentiation in K562 and Meg-01 cells. A radiation-induced thrombocytopenia mouse model was constructed to assess the therapeutic actions of AECRs on thrombocytopenia. Network pharmacology and experimental verification were carried out to clarify its mechanism against thrombocytopenia. RESULTS: AECRs promoted megakaryocyte differentiation in K562 and Meg-01 cells and accelerated platelet recovery and megakaryopoiesis with no systemic toxicity in radiation-induced thrombocytopenia mice. The PI3K/AKT, MEK/ERK, and JAK2/STAT3 signaling pathways contributed to AECR-induced megakaryocyte differentiation. The suppression of the above signaling pathways by their inhibitors blocked AERC-induced megakaryocyte differentiation. CONCLUSIONS: AECRs can promote megakaryopoiesis and thrombopoiesis through activating PI3K/AKT, MEK/ERK, and JAK2/STAT3 signaling pathways, which has the potential to treat radiation-induced thrombocytopenia in the clinic.

Deciphering transcriptome alterations in bone marrow hematopoiesis at single-cell resolution in immune thrombocytopenia.

Immune thrombocytopenia (ITP) is an autoimmune disorder, in which megakaryocyte dysfunction caused by an autoimmune reaction can lead to thrombocytopenia, although the underlying mechanisms remain unclear. Here, we performed single-cell transcriptome profiling of bone marrow CD34+ hematopoietic stem and progenitor cells (HSPCs) to determine defects in megakaryopoiesis in ITP. Gene expression, cell-cell interactions, and transcriptional regulatory networks varied in HSPCs of ITP, particularly in immune cell progenitors. Differentially expressed gene (DEG) analysis indicated that there was an impaired megakaryopoiesis of ITP. Flow cytometry confirmed that the number of CD9+ and HES1+ cells from Lin-CD34+CD45RA- HSPCs decreased in ITP. Liquid culture assays demonstrated that CD9+Lin-CD34+CD45RA- HSPCs tended to differentiate into megakaryocytes; however, this tendency was not observed in ITP patients and more erythrocytes were produced. The percentage of megakaryocytes differentiated from CD9+Lin-CD34+CD45RA- HSPCs was 3-fold higher than that of the CD9- counterparts from healthy controls (HCs), whereas, in ITP patients, the percentage decreased to only 1/4th of that in the HCs and was comparable to that from the CD9- HSPCs. Additionally, when co-cultured with pre-B cells from ITP patients, the differentiation of CD9+Lin-CD34+CD45RA- HSPCs toward the megakaryopoietic lineage was impaired. Further analysis revealed that megakaryocytic progenitors (MkP) can be divided into seven subclusters with different gene expression patterns and functions. The ITP-associated DEGs were MkP subtype-specific, with most DEGs concentrated in the subcluster possessing dual functions of immunomodulation and platelet generation. This study comprehensively dissects defective hematopoiesis and provides novel insights regarding the pathogenesis of ITP.

Bifunctional effect of the inflammatory cytokine tumor necrosis factor α on megakaryopoiesis and platelet production.

BACKGROUND AND OBJECTIVES: Platelets are affected by many factors, such as infectious or aseptic inflammation, and different inflammatory states may induce either thrombocytopenia or thrombocytosis. Tumor necrosis factor α (TNFα) is an important inflammatory cytokine that has been shown to affect the activity of hematopoietic stem cells. However, its role in megakaryocyte (MK) development and platelet production remains largely unknown. This study aimed to investigate the effects of TNFα on MK and platelet generation. METHODS AND RESULTS: The ex vivo study with human CD34+ cells demonstrated that TNFα differentially modulated commitment toward the MK lineage. Specifically, a low concentration of 0.5 ng/ml TNFα promoted MK maturation, proplatelet formation, and platelet production, whereas a high concentration of 10 ng/ml or more TNFα exhibited a substantial inhibitory effect on MK and platelet production. The distinct effect of TNFα on MKs was mainly dependent on TNFα receptor 1. TNFα differentially regulated the MAPK-ERK1/2 signaling pathway and the cytoskeletal proteins cofilin and MLC2. The in vivo study with Balb/c mice indicated that low-dose or high-dose TNFα administration differentially affected short-term platelet recovery after bone marrow transplantation. CONCLUSIONS: Our study revealed distinct roles for TNFα in megakaryopoiesis and thrombopoiesis and may provide new insights regarding the treatment for platelet disorders.

Age-restricted functional and developmental differences of neonatal platelets.

BACKGROUND: Developmental ontogeny of neonatal thrombopoiesis retains characteristics that are distinct from adults although molecular mechanisms remain unestablished. METHODS: We applied multiparameter quantitative platelet responses with integrated ribosome profiling/transcriptomic studies to better define gene/pathway perturbations regulating the neonatal-to-adult transition. A bioinformatics pipeline was developed to identify stable, neonatal-restricted platelet biomarkers for clinical application. RESULTS: Cord blood (CB) platelets retained the capacity for linear agonist-receptor coupling linked to phosphatidylserine (PS) exposure and α-granule release, although a restricted block in cross-agonist activation pathways was evident. Functional immaturity of synergistic signaling pathways was due to younger ontogenetic age and singular underdevelopment of the protein secretory gene network, with reciprocal expansion of developmental pathways (E2F, G2M checkpoint, c-Myc) important for megakaryocytopoiesis. Genetic perturbations regulating vesicle transport and fusion (TOM1L1, VAMP3, SNAP23, and DNM1L) and PS exposure and procoagulant activity (CLCN3) were the most significant, providing a molecular explanation for globally attenuated responses. Integrated transcriptomic and ribosomal footprints identified highly abundant (ribosome-protected) DEFA3 (encoding human defensin neutrophil peptide 3) and HBG1 as stable biomarkers of neonatal thrombopoiesis. Studies comparing CB- or adult-derived megakaryocytopoiesis confirmed inducible and abundant DEFA3 antigenic expression in CB megakaryocytes, ~3.5-fold greater than in leukocytes (the most abundant source in humans). An initial feasibility cohort of at-risk pregnancies manifested by maternal/fetal hemorrhage (chimerism) were applied for detection and validation of platelet HBG1 and DEFA3 as neonatal thrombopoiesis markers, most consistent for HBG1, which displayed gestational age-dependent expression. CONCLUSIONS: These studies establish an ontogenetically divergent stage of neonatal thrombopoiesis, and provide initial feasibility studies to track disordered fetal-to-adult megakaryocytopoiesis in vivo.

Relieving DYRK1A repression of MKL1 confers an adult-like phenotype to human infantile megakaryocytes.

Infantile (fetal and neonatal) megakaryocytes (Mks) have a distinct phenotype consisting of hyperproliferation, limited morphogenesis, and low platelet production capacity. These properties contribute to clinical problems that include thrombocytopenia in neonates, delayed platelet engraftment in recipients of cord blood stem cell transplants, and inefficient ex vivo platelet production from pluripotent stem cell-derived Mks. The infantile phenotype results from deficiency of the actin-regulated coactivator, MKL1, which programs cytoskeletal changes driving morphogenesis. As a strategy to complement this molecular defect, we screened pathways with the potential to affect MKL1 function and found that DYRK1A inhibition dramatically enhanced Mk morphogenesis in vitro and in vivo. Dyrk1 inhibitors rescued enlargement, polyploidization, and thrombopoiesis in human neonatal Mks. Mks derived from induced pluripotent stem cells responded in a similar manner. Progenitors undergoing Dyrk1 inhibition demonstrated filamentous actin assembly, MKL1 nuclear translocation, and modulation of MKL1 target genes. Loss-of-function studies confirmed MKL1 involvement in this morphogenetic pathway. Expression of Ablim2, a stabilizer of filamentous actin, increased with Dyrk1 inhibition, and Ablim2 knockdown abrogated the actin, MKL1, and morphogenetic responses to Dyrk1 inhibition. These results delineate a pharmacologically tractable morphogenetic pathway whose manipulation may alleviate clinical problems associated with the limited thrombopoietic capacity of infantile Mks.

Fate mapping of hematopoietic stem cells reveals two pathways of native thrombopoiesis.

Hematopoietic stem cells (HSCs) produce highly diverse cell lineages. Here, we chart native lineage pathways emanating from HSCs and define their physiological regulation by computationally integrating experimental approaches for fate mapping, mitotic tracking, and single-cell RNA sequencing. We find that lineages begin to split when cells leave the tip HSC population, marked by high Sca-1 and CD201 expression. Downstream, HSCs either retain high Sca-1 expression and the ability to generate lymphocytes, or irreversibly reduce Sca-1 level and enter into erythro-myelopoiesis or thrombopoiesis. Thrombopoiesis is the sum of two pathways that make comparable contributions in steady state, a long route via multipotent progenitors and CD48hi megakaryocyte progenitors (MkPs), and a short route from HSCs to developmentally distinct CD48-/lo MkPs. Enhanced thrombopoietin signaling differentially accelerates the short pathway, enabling a rapid response to increasing demand. In sum, we provide a blueprint for mapping physiological differentiation fluxes from HSCs and decipher two functionally distinct pathways of native thrombopoiesis.

Optimization of human platelet lysate production and pathogen reduction in a public blood transfusion center.

BACKGROUND: Human platelet lysate (HPL) has been proposed as a safe and efficient xeno-free alternative to fetal bovine serum (FBS) for large-scale culturing of cell-based medicinal products. However, the use of blood derivatives poses a potential risk of pathogen transmission. To mitigate this risk, different pathogen reduction treatment (PRT) practices can be applied on starting materials or on final products, but these methods might modify the final composition and the quality of the products. STUDY DESIGN AND METHODS: We evaluated the impact of applying a PRT based on riboflavin and ultraviolet irradiation on the raw materials used to manufacture an improved Good Manufacturing Practices (GMP)-grade HPL product in a public blood center. Growth promotion and the levels of growth factors and proteins were compared between an inactivated product (HPL4-i) and a non-inactivated product (HPL4). Stability studies were performed at 4°C, -20°C, and -80°C. RESULTS: The application of a PRT on the starting materials significantly altered the protein composition of HPL4-i as compared with HPL4. Despite this, the growth promoting rates were unaffected when compared with FBS used as a control. While all products were stable at -20°C and -80°C for 24 months, a significant decrease in the activity of HPL4-i was observed when stored at 4°C. CONCLUSION: Our results show that the application of a PRT based on riboflavin and ultraviolet light on starting materials used in the manufacture of HPL modifies the final composition of the product, yet its cell growth promoting activity is maintained at levels similar to those of non-inactivated products.