Erythropoiesis and Megakaryopoiesis in a Dish

Erythrocytes and platelets are the major cellular components of blood. Several hereditary diseases affect the production/stability of red blood cells (RBCs) and platelets (Plts) resulting in anemia or bleeding, respectively. Patients with such disorders may require recurrent transfusions, which bear a risk to develop alloantibodies and ultimately may result in transfusion product refractoriness. Cell culture models enable to unravel disease mechanisms, and to screen for alternative therapeutic products. Besides these applications, the ultimate goal is the large-scale production of blood effector cells for transfusion. Cultured RBCs that lack many of the common blood group antigens and Plts-lacking HLA expression would improve transfusion practice. Large numbers of RBCs and Plts can already be generated using hematopoietic stem cells derived from fetal liver, cord blood, peripheral blood, and bone marrow as starting material for cell culture. The recent advances to generate blood cells from induced pluripotent stem cells provide a donor-independent, immortal primary source for cell culture models. This enables us to study developmental switches during erythropoiesis/megakaryopoiesis and provides potential future therapeutic applications. In this review, we will discuss how erythropoiesis and megakaryopoiesis are mimicked in culture systems and how these models relate to the in vivo process

Understanding and Improving Platelet-Delivered Factor (F) VIII Hemostatic Efficacy in Hemophilia A Mice

Hemophilia A is the most common, inherited severe bleeding diathesis and is due to a deficiency of functional Factor (F) VIII. Two challenges to current FVIII replacement therapy are maintaining sustained FVIII corrections and treating the 30% population of Hemophilia A patients who develop FVIII inhibitors. Gene therapy promises to be an attractive treatment for hemophilia A as there is a wide therapeutic window for FVIII corrective plasma levels. Since platelets target to hemostatic injuries, ectopically expressed platelet (p) FVIII is an attractive alternative FVIII delivery strategy. We have already shown the effectiveness of platelet human B-domainless factor VIII (phBF8) in the presence of circulating inhibitors in FVIIInull mice. However, the clots formed are unstable and lead to increased embolization with phBF8. This dissertation describes platelet-specific gene therapy using three FVIII variants with enhanced activity: inactivation resistant FVIII (IR8), canine (c) B-domainless FVIII (cBF8), and hBF8R1645H. Each improved clotting efficacy in FVIIInull mice and decreased embolic risk though to different degrees. For example, we found that despite pcBF8 having ~30% antigenic levels of phBF8, it corrected clot instability seen in a cremaster injury model in FVIIInull mice. Since IR8 does not bind FVIII carrier protein, Von Willebrand Factor (vWF), efficiently, we tested the effectiveness of this variant in the presence of circulating FVIII inhibitors, and found that vWF binding was not needed for IR8 to be effective. To understand the cellular mechanism for decreased cBF8 relative to hBF8 expression level in megakaryocytes, we studied mRNA levels and the effect of pcBF8 on megakaryopoiesis and found that the level of cBF8 is related to increased cellular apoptosis. Finally to understand the mechanistic basis of pcBF8 increased hemostatic effectiveness, we examined whether mutation of the cleavage site for the Golgi apparatus enzyme PACE/furin in hBF8 to that of cBF8, which increases the FVIII’s activity, would also enhance hBF8R1645H pFVIII activity and affirm that this occurs. These studies provide new insights into pFVIII during megakaryopoiesis and how to optimize pFVIII hemostatic efficacy that should be useful in translating this therapeutic model to a large animal hemophilia A model and future clinical trials

New functions of platelet C3G: Involvement in TPO-regulation, ischemia-induced angiogenesis and tumor metastasis

[ES] Las GTPasas son proteínas que regulan una gran variedad de procesos celulares, entre los que cabe destacar la proliferación, la diferenciación celular y la apoptosis. Estas proteínas alternan entre dos confirmaciones: una activa o unida a GTP y una inactiva o unida a GDP. El intercambio de GDP por GTP esta catalizado por un grupo de proteínas denominadas GEF (factores intercambiadores de nucleótidos de guanina), mientras que las proteínas GAP (proteínas activadoras de la actividad GTPasa) inhiben a la GTPasa. C3G es un GEF para varias GTPasas de la familia de Ras, principalmente de Rap1, R-Ras y TC21, y para una GTPasa de la familia de Rho, TC10. Mediante el uso de modelos animales que expresan de manera específica en plaquetas y megacariocitos o bien C3G (tgC3G), o bien una forma mutante de C3G (caracterizada por la pérdida del dominio catalítico, tgC3GCat), nuestro grupo ha demostrado la participación de C3G en la diferenciación megacariocítica, así como en la regulación de la función hemostática de las plaquetas. En concreto, las plaquetas tgC3G presentan una mayor activación y agregación plaquetaria, que se correlaciona con tiempos de sangrado significativamente inferiores en los ratones tgC3G, además de un incremento en la formación de trombos en modelos in vivo. La sobreexpresión de C3G plaquetario, también genera una alteración en la secreción de los gránulos-α, caracterizada por la retención del factor de crecimiento del endotelio vascular (VEGF) y de trombospondina- 1 (TSP-1) en el citoplasma de las plaquetas, dando lugar a un secretoma netamente proangiogénico. Como resultado de la mayor capacidad proangiogénica de las plaquetas que sobreexpresan C3G, los ratones tgC3G mostraron un crecimiento tumoral más rápido en dos modelos heterotópicos de implantación tumoral. Además, la proteína C3G plaquetaria promueve la metástasis pulmonar de células de melanoma (B16-F10). Sin embargo, la expresión transgénica de C3G no altera los recuentos plaquetarios en sangre periférica. En esta Tesis, hemos profundizado en el papel de C3G en la megacariopoyesis, en la angiogénesis inducida por isquemia y en la metástasis tumoral. Para ello, hemos desarrollado un modelo animal adicional (C3G-KO), en el cual C3G se encuentra específicamente delecionado en megacariocitos (Mk). Al igual que lo observado en ratones tgC3G, los animales C3G-KO tampoco mostraron diferencias ni en el número de Mk en médula ósea, ni en los recuentos plaquetarios en sangre periférica. Sin embargo, la deleción de C3G resultó en una mayor maduración megacariocítica in vitro cuando las médulas óseas fueron cultivas en medio enriquecido con trombopoyetina (TPO) junto con un cocktail de citocinas, sugiriendo un posible papel de C3G en una megacariopoyesis patológica. En base a esto, hemos analizamos el papel de C3G en dos modelos in vivo de megacariopoyesis patológica: la inyección de TPO y la mielosupresión inducida por 5- Fluoruracilo (5-FU). La inyección intravenosa de TPO estimula la megacariopoyesis, incrementando los niveles plaquetarios; mientras que el 5-FU induce la depleción de la médula ósea alrededor del séptimo día tas la inyección, lo que va seguido de un profundo incremento en el recuento plaquetario, proceso conocido como rebote plaquetario (platelet rebound) tras 10-15 días de tratamiento

Caracterización de nuevos genes y variantes implicados en Trastornos Plaquetarios Congénitos: de los datos genómicos a los estudios funcionales

Tesis por compendio de publicaciones[ES] El objetivo general de este estudio es: Caracterizar el fenotipo clínico y plaquetario asociado a las alteraciones moleculares encontradas por secuenciación de alto rendimiento en genes implicados en los Trastornos Plaquetarios Congénitos, y demostrar el mecanismo relacionado con la patogenicidad utilizando cultivos de megacariocitos in vitro y ex vivo, y modelos de enfermedad in vivo generados por la tecnología CRISPR/Cas9. Como objetivos específicos planteados, se encuentran: • Caracterización funcional de la variante RUNX1 p.Leu56Ser, encontrada previamente por un panel de genes de diseño propio, utilizando una aproximación in vivo. o Generar un modelo murino knock-in portador de la variante RUNX1 p.Leu43Ser (que imita a la variante en línea germinal humana p.Leu56Ser) mediante CRISPR/Cas9, para establecer su implicación en el trastorno plaquetario. o Conocer las vías de señalización y los determinantes biológicos que subyacen a la progresión leucémica asociada a las variantes de RUNX1. • Aplicación de la secuenciación del exoma completo para la identificación de nuevos genes implicados en los Trastornos Plaquetarios Congénitos, y evaluación de los mecanismos fisiopatológicos que originan la enfermedad mediante modelos funcionales in vivo y ex vivo. o Analizar el fenotipo clínico y de laboratorio de la tercera familia en el mundo con trombocitopenia asociada a variantes en TPM4, y caracterizar en profundidad su papel en la remodelación del citoesqueleto plaquetario. o Evaluar el fenotipo clínico y plaquetario de tres pacientes de dos familias no relacionadas con macrotrombocitopenia sindrómica asociada a variantes en heterocigosis compuesta en GALE. o Investigar el efecto funcional de las variantes genéticas identificadas en GALE durante la megacariopoyesis y la trombopoyesis, y su papel en la glicosilación de los megacariocitos y las plaquetas utilizando un modelo de sobreexpresión in vitro y un cultivo ex vivo de megacariocitos de pacientes

Monitoring and mathematical modeling of in vitro human megakaryocyte expansion and maturation dynamics

La mégakaryopoïèse est un processus complexe, qui prend naissance à partir des cellules souches hématopoïétiques (HSC). Ces dernières se différencient par étapes successives en mégakaryocytes (MKs) qui, suite à leur maturation, libèrent les plaquettes. Afin de modéliser le sort des HSCs lors de la mégakaryopoïèse en culture, un nouveau modèle mathématique a été développé, basé sur un programme de différenciation tridimensionnelle (3-D) où chaque sous-population est représentée par un compartiment. Dans le but d’évaluer la prolifération, la différenciation des MKs immatures puis matures, la cinétique de mort cellulaire ainsi que le nombre de plaquettes produites, à partir des cellules de sang de cordon (CB) ombilical enrichies en CD34+, un ensemble d'équations différentielles a été déployé. Les cellules CD34+ ont été placées en culture dans un milieu optimisé pour la différenciation mégakaryocytaire. Les paramètres cinétiques ont été estimés pour deux températures d'incubation (37°C versus 39°C). Les résultats des régressions ont été validés par l'évaluation de l'estimabilité des paramètres, en utilisant des analyses de sensibilité locale et globale, puis la détermination d'un intervalle de confiance. Ceux-ci ont été comparés par le biais de tests statistiques et d’analyses en composante principale (ACP). Le modèle proposé pourrait permettre de mieux comprendre les phénomènes complexes observés. Les MKs sont uniques parmi les cellules hématopoïétiques, étant les seules à devenir polyploïdes au cours de leur développement par l’entremise de l’endomitose, un processus mitotique qui se termine prématurément durant la cytocinèse. Pour obtenir une image plus complète et exhaustive de la mégacaryopoïèse, une approche d’imagerie cellulaire à grand champ et à long terme a été développée permettant de suivre individuellement l'évolution des HSCs lors de leur différenciation ex vivo. Cela a permis de démontrer que les MKs polyploïdes sont encore capables de se diviser et de produire des cellules filles polyploïdes, et que ce processus est plus fréquent chez les MKs issues de CB que de moelle osseuse d'adulte. De plus, le processus de formation des proplaquettes semble également réversible. Les phénomènes énoncés plus haut étaient inversement proportionnels au niveau de ploïdie des MKs. En conclusion, cette étude a dévoilé de nouvelles propriétés jusqu’ici inconnues des MKs.Megakaryopoiesis is a complex process, which is initiated with the proliferation and the differentiation of hematopoietic stem cells (HSC) into megakaryocytes (MK), followed by the maturation of MK and ended by platelet release. To describe the fates of HSC during ex vivo megakaryopoiesis, a new mathematical model was developed based on a 3-dimensional kinetic developmental program. To address this, a set of differential equations was applied to analyze the proliferation, differentiation and death kinetic rates of purified cord blood (CB)-CD34+ cells, immature and mature MKs, as well as platelet number and productivity. CB-CD34+ cells were placed in culture optimized for MK differentiation. The kinetic parameters were estimated for two incubation temperatures (37°C vs. 39°C). The regression results have been validated by assessing the parameter identifiability using local and global sensitivity analyses and confidence intervals, and compared using statistical tests and principal component analysis (PCA). Furthermore, PCA was applied on the solution matrix to construct a simplified MK differentiation pathway model, and to reveal dependencies among the model parameters. The proposed model provides insight into phenomena that would be otherwise difficult to interpret. MKs are unique among mammalian marrow cells as they polyploidize during their natural development. It is universally accepted that MK becomes polyploid by repeatedly deviating from normal cell cycling, where it ceases to complete cytokinesis and divide. To challenge this long-standing hypothesis and to obtain a more comprehensive picture of megakaryopoiesis, a long-term and large-field live cell imaging approach of in vitro MK culture was developed. Using CB- and bone marrow (BM)-CD34+ as starting cells, the direct observation of cells undergoing differentiation and maturation over a 5-day culture period is reported for the first time. Herein, direct visual proof that polyploid MKs can complete cytokinesis during its normal development is presented. This phenomenon was found not restricted to CB- as the BM-derived polyploid MK also underwent division. However the latter showed significantly lower proliferation rate. This new finding explains in part the unresolved issue of low ploidy levels observed in CB-MK and contests the notion that polyploid MKs do not divide

The EHA Research Roadmap: Normal Hematopoiesis.

International audienceIn 2016, the European Hematology Association (EHA) published the EHA Roadmap for European Hematology Research1 aiming to highlight achievements in the diagnostics and treatment of blood disorders, and to better inform European policy makers and other stakeholders about the urgent clinical and scientific needs and priorities in the field of hematology. Each section was coordinated by 1–2 section editors who were leading international experts in the field. In the 5 years that have followed, advances in the field of hematology have been plentiful. As such, EHA is pleased to present an updated Research Roadmap, now including 11 sections, each of which will be published separately. The updated EHA Research Roadmap identifies the most urgent priorities in hematology research and clinical science, therefore supporting a more informed, focused, and ideally a more funded future for European hematology research. The 11 EHA Research Roadmap sections include Normal Hematopoiesis; Malignant Lymphoid Diseases; Malignant Myeloid Diseases; Anemias and Related Diseases; Platelet Disorders; Blood Coagulation and Hemostatic Disorders; Transfusion Medicine; Infections in Hematology; Hematopoietic Stem Cell Transplantation; CAR-T and Other Cell-based Immune Therapies; and Gene Therapy

Functional hyper-IL-6 from vaccinia virus-colonized tumors triggers platelet formation and helps to alleviate toxicity of mitomycin C enhanced virus therapy

<p>Abstract</p> <p>Background</p> <p>Combination of oncolytic vaccinia virus therapy with conventional chemotherapy has shown promise for tumor therapy. However, side effects of chemotherapy including thrombocytopenia, still remain problematic.</p> <p>Methods</p> <p>Here, we describe a novel approach to optimize combination therapy of oncolytic virus and chemotherapy utilizing virus-encoding hyper-IL-6, GLV-1h90, to reduce chemotherapy-associated side effects.</p> <p>Results</p> <p>We showed that the hyper-IL-6 cytokine was successfully produced by GLV-1h90 and was functional both in cell culture as well as in tumor-bearing animals, in which the cytokine-producing vaccinia virus strain was well tolerated. When combined with the chemotherapeutic mitomycin C, the anti-tumor effect of the oncolytic virotherapy was significantly enhanced. Moreover, hyper-IL-6 expression greatly reduced the time interval during which the mice suffered from chemotherapy-induced thrombocytopenia.</p> <p>Conclusion</p> <p>Therefore, future clinical application would benefit from careful investigation of additional cytokine treatment to reduce chemotherapy-induced side effects.</p

Fli1 Expression Levels During Megakaryopoiesis Affect Thrombopoiesis And Platelet Biology

Friend Leukemia Virus Integration 1 (FLI1) is a critical transcription factor (TF) in terminal megakaryocyte differentiation. It is amongst the genes missing from an inherited hemizygous deletion on chromosome 11q termed Jacobsen syndrome and often results in a dysmegakaryopoiesis and macrothrombocytopenia termed Paris Trousseau syndrome (PTSx) described as being due to FLI1 allelic exclusion. It has also been reported that heterozygote FLI1 mutations in its DNA-binding domain region cause thrombocytopenia in patients suspected to have inherited platelet defects. To date, there are no reports containing comprehensive in vitro or in vivo characterization of platelet defects due to heterozygous FLI1 deletion or mutations, or that of platelets expressing increased levels of FLI1. We used induced pluripotent stem cell (iPSC)- derived megakaryocytes (iMegs) to determine if the platelet disorder observed in PTSx could be replicated, either with iPSCs generated from a PTSx patient or from a targeted heterozygous knockout of FLI1 (FLI1+/-) in control iPSCs. These studies indicate that PTSx and FLI1+/- iMegs replicate many of the clinical features described in PTSx and showed more in vitro injury to the resulting iMegs with fewer platelets released in vivo. These platelets had shortened half-lives and were functionally defective. We then examined whether increased levels of FLI1 would affect megakaryopoiesis and thrombopoiesis, and found an increased number of iMegs with less in vitro injury compared to control iMegs. FLI1-overexpressing iMegs also released more platelets in recipient mice with increased half-life and functionality. These studies confirm FLI1 heterozygosity results in defects in megakaryopoiesis and thrombopoiesis similar to that noted with other megakaryocyte-specific TFs, but unlike those TFs, FLI1 overexpression is not associated with quality or quantitative platelet deficiencies, but improved yield and functionality that may have clinical applicability

A novel nonsense variant in TPM4 caused dominant macrothrombocytopenia, mild bleeding tendency and disrupted cytoskeleton remodeling

[Background]: Rare inherited thrombocytopenias are caused by alterations in genes involved in megakaryopoiesis, thrombopoiesis and/or platelet release. Diagnosis is challenging due to poor specificity of platelet laboratory assays, large numbers of culprit genes, and difficult assessment of the pathogenicity of novel variants. [Objectives]: To characterize the clinical and laboratory phenotype, and identifying the underlying molecular alteration, in a pedigree with thrombocytopenia of uncertain etiology. [Patients/Methods]: Index case was enrolled in our Spanish multicentric project of inherited platelet disorders due to lifelong thrombocytopenia and bleeding. Bleeding score was recorded by ISTH‐BAT. Laboratory phenotyping consisted of blood cells count, blood film, platelet aggregation and flow cytometric analysis. Genotyping was made by whole‐exome sequencing (WES). Cytoskeleton proteins were analyzed in resting/spreading platelets by immunofluorescence and immunoblotting. [Results]: Five family members displayed lifelong mild thrombocytopenia with a high number of enlarged platelets in blood film, and mild bleeding tendency. Patient's platelets showed normal aggregation and granule secretion response to several agonists. WES revealed a novel nonsense variant (c.322C>T; p.Gln108*) in TPM4 (NM_003290.3), the gene encoding for tropomyosin‐4 (TPM4). This variant led to impairment of platelet spreading capacity after stimulation with TRAP‐6 and CRP, delocalization of TPM4 in activated platelets, and significantly reduced TPM4 levels in platelet lysates. Moreover, the index case displayed up‐regulation of TPM2 and TPM3 mRNA levels. [Conclusions]: This study identifies a novel TPM4 nonsense variant segregating with macrothrombocytopenia and impaired platelet cytoskeletal remodeling and spreading. These findings support the relevant role of TPM4 in thrombopoiesis and further expand our knowledge of TPM4‐related thrombocytopenia.This work was partially supported by grants from Instituto de Salud Carlos III (ISCIII) and Feder (PI17/01966, PI20/00926), Gerencia Regional de Salud (GRS2061A/19, GRS2135/A/2020, GRS2314/A/2021), Fundación Mutua Madrileña (FMM, AP172142019) and Sociedad Española de Trombosis y Hemostasia (SETHFETH; Premio López Borrasca 2019 and Ayuda a Grupos de Trabajo en Patología Hemorrágica 2020 and 2021).Peer reviewe

Generation and Characterization of a Knock-In Allele of EKLF: Probing the in vivo Role of the Chromatin Remodeling Domain in Definitive Hematopoietic Cells

The zinc finger-encoding transacting factor EKLF, or erythroid Krüppel-like factor, binds key regulatory elements of many erythroid-specific genes, and is essential for definitive erythropoiesis. Mice lacking this factor die of anemia by E15.5 of gestation, failing to activate β-globin gene transcription, and demonstrating a block in the erythroid differentiation program at the primitive erythroblast stage. In contrast, megakaryocytic progenitors are amplified in EKLF-null embryos, with increased Fli-1 gene expression, a marker of early megakaryocytic differentiation. These observations are consistent with the idea that EKLF modulates the megakaryocytic-erythroid (M-E) differentiation switch. Our laboratory has previously demonstrated that an amino terminal sequence of EKLF (D221EKLF) is required to induce chromatin remodeling at the β-globin promoter in an EKLF-null erythroid cell line. However, additional amino terminal sequences are required for initiation of β-globin gene transcription. To evaluate the role of this chromatin remodeling domain in erythroid and megakaryocytic differentiation in vivo, I have generated a knock-in allele of D221EKLF. Using the recombineering method, a lambda phage-based homolgous recombination method in E. coli, cDNA encoding theD221EKLF domain has been inserted into the endogenous initiation site, thus placing the mutant protein under the cis-regulatory elements of the endogenous murine EKLF locus. Subsequently, D221EKLF alleles have been generated by gene targeting in ES cells. I have used the mice to probe the in vivo role of D221EKLF in definitive hematopoietic cells. Similar to EKLF-null embryos, mice homozygous for the D221EKLF mutant allele die of anemia by E15.5 of gestation. Molecular analysis ofD221EKLF erythroblasts reveals i) a failure to activate β-globin gene transcription; ii) lack of GATA-1 and NF-E2 recruitment to the β-globin promoter; iii) a block in terminal erythroid differentiation. In contrast to erythroid cells lacking EKLF, D221EKLF erythroid progenitors demonstrate appropriate binding of the D221EKLF encoding domain to all EKLF-regulatory sequences and a chromatin architecture and histone modification pattern at erythroid-specific genes that recapitulate the events observed in wild-type EKLF erythroblasts at a similar stage of erythroid ontogeny. Examining the role of D221EKLF in megakaryopoiesis, I observed inhibition of megakaryocytic progenitor expansion in D221EKLF fetal hematopoietic cell populations when compared to EKLF-null embryos. Molecular analysis of D221EKLF erythroblasts reveals i) binding of theD221EKLF mutant protein to the Fli-1 promoter with inhibition of gene transcription; ii) hypoacetylation of histone H3 at the Fli-1 promoter; iii) recruitment of a Sin3A-containing corepressor complex to the Fli-1 promoter. Taken together, my results suggest strongly that the unique D221EKLF domain is sufficient to modulate the chromatin-specific roles of EKLF at erythroid- and megakaryocytic-specific loci in definitive hematopoietic cells in vivo