RESUMEN
Current research in the field of transfusion medicine is focused on developing innovative approaches to generate populations of functional megakaryocytes (MKs) ex vivo. This may open perspectives to establish alternative therapies for donor platelet transfusion in the management of thrombocytopenic patients and pave the way for novel regenerative approaches. Efficient cryopreservation techniques can provide the opportunity for long-term storage and accumulation of necessary amounts of MKs in a ready-to-use manner. However, in this case, besides the viability, it is crucial to consider the recovery of functional MK properties after the impact of freezing. In this chapter, the possibility to cryopreserve iPSC-derived MKs is described. In particular, the methods for a comprehensive analysis of phenotypic and functional features of MKs after cryopreservation are proposed. The use of cryopreserved in vitro-produced MKs may benefit to the field of transfusion medicine to overcome the lack of sufficient blood donors.
Asunto(s)
Plaquetas/citología , Técnicas de Cultivo de Célula/métodos , Separación Celular/métodos , Criopreservación/métodos , Crioprotectores/farmacología , Células Madre Pluripotentes Inducidas/citología , Megacariocitos/citología , Animales , Plaquetas/efectos de los fármacos , Diferenciación Celular , Humanos , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Megacariocitos/efectos de los fármacosRESUMEN
Huangqi, the dried root of Radix Astragali, is an essential herb in Traditional Chinese Medicine and has been used to promote hematopoiesis for centuries. Astragalus polysaccharide (ASPS), the bioactive compound of Huangqi, serves a crucial role in hematopoiesis. The aim of the present study was to investigate the hematopoietic effects, in particular the thrombopoietic effects, and the molecular mechanisms of ASPS using an irradiationinduced myelosuppressive mouse model. Colonyforming unit assays, flow cytometric analysis of apoptosis, ELISAs, Giemsa staining and western blotting were performed to determine the hematopoietic and antiapoptotic effects of ASPS. The results demonstrated that ASPS enhanced the recovery of red blood cells at day 21 following treatment, as well as platelets and white blood cells at day 14. In addition, ASPS promoted colony formation in all lineages (megakaryocytes, granulocyte monocytes, erythroid cells and fibroblasts). The morphological study of the bone marrow demonstrated that trilineage hematopoiesis was preserved in the ASPS and thrombopoietin (TPO)treated groups compared with the control group. The overall cellularity (mean total cell count/area) of the ASPStreated group was similar to that of the TPOtreated group. Additionally, in vitro experiments indicated that treatment with 100 µg/ml ASPS exhibited the maximum effect on colony formation. ASPS attenuated cell apoptosis in megakaryocytic cells via inhibiting the mitochondrial caspase3 signaling pathway. In conclusion, ASPS promoted hematopoiesis in irradiated myelosuppressive mice possibly via enhancing hematopoietic stem/progenitor cell proliferation and inhibiting megakaryocytes apoptosis.
Asunto(s)
Medicamentos Herbarios Chinos/química , Megacariocitos/citología , Polisacáridos/administración & dosificación , Traumatismos Experimentales por Radiación/tratamiento farmacológico , Trombocitopenia/prevención & control , Animales , Apoptosis/efectos de los fármacos , Astragalus propinquus , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Hematopoyesis/efectos de los fármacos , Hematopoyesis/efectos de la radiación , Inyecciones Intraperitoneales , Masculino , Megacariocitos/efectos de los fármacos , Megacariocitos/efectos de la radiación , Ratones , Polisacáridos/farmacología , Traumatismos Experimentales por Radiación/complicaciones , Traumatismos Experimentales por Radiación/metabolismo , Trombocitopenia/etiologíaRESUMEN
During maturation, megakaryocytes (MKs) express ß1-tubulin (TUBB1) and rearrange their microtubule components to enlarge, form proplatelets, and eventually release platelets. The development of a platform to identify in vitro conditions that would efficiently promote MK development could potentially enable large-scale platelet production. Here, we show that an immortalized MK cell line (imMKCL) genetically modified to express the ß1-tubulin-Venus reporter provides a practical system to efficiently monitor the in vitro production of platelet-like particles (PLPs). The Venus transgene was inserted downstream of the TUBB1 locus in imMKCLs using CRISPR/Cas9, and the expression was visualized by Venus fluorescence intensity. This imMKCL reporter line was then used for high-throughput drug screening. We identified several compounds that significantly improved the efficiency of PLP production in vitro under feeder-free conditions and showed a significant tendency to recover platelets in vivo in a mouse thrombocytopenia model induced by anti-GPIbα antibody administration. Interestingly, most of these compounds, including a WNT signaling pathway inhibitor, Wnt-C59, antagonized the aryl hydrocarbon receptor (AhR) to increase PLP production, confirming the crucial role of AhR inhibition in MK maturation. Consistently, small interfering RNA treatment against AhR increased the Venus intensity and PLP production. TCS 359, an FLT3 inhibitor, significantly increased PLP production independently of FLT3 or AhR. This study highlights the usefulness of the ß1-tubulin reporter MK line as a useful tool to study the mechanisms underlying thrombopoiesis and to identify novel inducers of ex vivo platelet production.
Asunto(s)
Plaquetas/citología , Descubrimiento de Drogas/métodos , Genes Reporteros/genética , Megacariocitos/metabolismo , Tubulina (Proteína)/metabolismo , Animales , Línea Celular , Evaluación Preclínica de Medicamentos/métodos , Humanos , Células Madre Pluripotentes Inducidas/citología , Luciferasas/genética , Masculino , Megacariocitos/citología , Ratones , Ratones Endogámicos C57BL , Receptores de Hidrocarburo de Aril/metabolismo , TrombopoyesisRESUMEN
Megakaryocytopoiesis results in the formation of platelets, which are essential for hemostasis. Decreased production or increased destruction of platelets can cause thrombocytopenia, in which platelet transfusion is the mode of treatment. The present study is aimed in generation of megakaryocytes (MKs) and platelet from human hematopoietic stem cells (HSCs). The purity of HSCs was assessed through Flow cytometry and immunocytochemistry (ICC) studies. These pure HSCs were induced with thrombopoietin (TPO), similarly with Andrographis paniculata extract (APE) for 21 days to generate MKs. The APE is mainly composed of andrographolide which stimulates TPO from the liver, and this binds to CD110 present on the surface of HSCs and triggers the proliferation of HSCs and initiate higher MKs population subsequently, a large number of platelets. The results of the present study showed increased proliferation of HSCs grown in the presence of APE and revealed a high population of CD41a and CD42b positive MKs as enumerated by Flow cytometry compared with TPO induced MKs. These results also concurred with qRT-PCR and western blot analysis. The scanning electron microscopy (SEM) revealed the morphology of differentiated MKs and platelets were similar to human blood platelets. The differentiated MKs in APE exhibited polyploidy up to 32â¯N while TPO induced MKs showed polyploidy of 8â¯N, these results corroborated with colony forming unit assay. On thrombin stimulation, high expression of P-selectin (CD62p) and fibrinogen binding were detected in APE induced platelets. Autologous transplantation of platelets generated from APE may be a useful option in thrombocytopenia condition.
Asunto(s)
Plaquetas/citología , Diferenciación Celular , Células Madre Hematopoyéticas/citología , Megacariocitos/citología , Andrographis paniculata , Células Cultivadas , Citometría de Flujo , Expresión Génica/efectos de los fármacos , Humanos , Megacariocitos/metabolismo , Megacariocitos/ultraestructura , Microscopía Electrónica de Rastreo , Extractos Vegetales/farmacología , Trombopoyesis/efectos de los fármacos , Trombopoyesis/genética , Trombopoyetina/farmacologíaRESUMEN
BACKGROUND: Iron deficiency anemia (IDA) is characterized by depletion of total body iron stores or a poor supply of plasma iron. By contrast, chronic inflammation makes iron unavailable for hematopoiesis through a cytokinemediated cascade and leads to a condition known as anemia of chronic disease (AOC). However, the laboratory data regarding the regulatory role of iron metabolism on platelet count has not been fully discussed yet. In this study, we investigated the relationship between iron status and platelet production according to different anemic mechanisms representing different iron metabolisms. METHODS: The study included a total of 759 specimens. Blood samples were obtained through venipuncture. The complete blood count was measured using an Advia 2120 (Siemens Healthcare Diagnostics Inc., USA). Biochemical indices including iron level were estimated using a Toshiba chemical analyzer (Toshiba, Japan). RESULTS: In the AOC group, we found a significant relationship between platelet count and serum iron level (p < 0.27), whereas there was no correlation in the IDA group. Moreover, when the AOC patient group was subdivided by serum iron level, a remarkable difference was observed as follows. The platelet count was significantly correlated with serum iron level only in the AOC group with decreased serum iron levels (serum iron < 50 µg/dL) (p < 0.0001), while there was no correlation in the AOC group with normal serum iron levels (serum iron 50 - 100 µg/dL). CONCLUSIONS: Iron deficiency in AOC involves upregulated hepcidin production induced by elevated inflammatory cytokines. This can cause increased iron sequestration in macrophages and decreased iron absorption for bone marrow. The condition of decreased megakaryocytic iron supply makes megakaryocytes with higher ploidy which can release more platelets than lower ploidy. Moreover, reactive thrombocytosis in inflammatory states occurs by cytokine cascades involving interleukin 6 and thrombopoietin in AOC. These two features may enhance thrombocytosis in patients of AOC with decreased iron level. In the future, further study should be performed to elucidate regulating mechanism of iron metabolism for megakaryopoiesis in AOC patients, and guide proper supplemental therapy of iron to decrease thrombotic risk due to reactive thrombocytosis in various kinds of anemia.
Asunto(s)
Anemia/sangre , Hierro/sangre , Megacariocitos/metabolismo , Trombopoyesis , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Anemia Ferropénica/sangre , Enfermedad Crónica , Citocinas/sangre , Femenino , Humanos , Hierro/metabolismo , Masculino , Megacariocitos/citología , Persona de Mediana Edad , Recuento de Plaquetas , Adulto JovenRESUMEN
BACKGROUND: Hepatoprotective activity along with improved survival percentage and hematological parameters prior to whole body irradiation were reported with Justicia adhatoda extracts. PURPOSE: To evaluate the thrombopoietic potential of Justicia adhatoda L. leaf extract in megakaryocyte differentiation METHODS: Ethanol extracts were prepared using soxhlet extraction method, and IC50 value was determined. The effect of ethanol extracts obtained from Justicia adhatoda on megakaryocyte maturation and development in megakaryocytic Dami cell lines was tested. Expression of megakaryocyte specific markers, CD61 and CD41, were assessed using flow cytometry and fluorescence microscopy. In addition, cell cycle analysis and mitochondrial membrane potential were analyzed by flow cytometry. Gene expression analysis was performed using qRT-PCR. RESULTS: At a concentration of 40⯵g/ml, the leaf extracts of Justicia adhatoda for 72â¯h induced the megakaryocytic features in megakaryocytic Dami cell lines. The megakaryocyte specific markers, CD41 and CD61, were up-regulated (2.2 and 12.4 fold, respectively), and more number of cells entered into synthetic (S) and G2/M phase as compared with untreated cell (23.1% vs 16.6% and 70.2% vs 42.3%, respectively) showing maturation. RUNX1 (a transcription factor essential for embryonic hematopoiesis and adult megkaryocyte maturation) and c-Mpl (the receptor for TPO) were upregulated, and the suppressor of cytokine signaling (SOCS) 1 and SOCS3 were down-regulated upon treatment with Justicia adhatoda. Justicia adhatoda enhanced mitochondrial ROS generation by 28-fold, increased the permeability of mitochondrial membrane and showed an inverse correlation in superoxide dismutase levels. CONCLUSION: Justicia adhatoda could enhance mitochondrial ROS generation and increase the permeability of mitochondrial membrane, thereby inducing megakaryocytic maturation. Our findings suggest thrombopoietic potential of Justicia adhatoda leaf extract on megakaryocyte differentiation.
Asunto(s)
Género Justicia/química , Megacariocitos/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Extractos Vegetales/farmacología , Especies Reactivas de Oxígeno/metabolismo , Diferenciación Celular/efectos de los fármacos , Línea Celular , Subunidad alfa 2 del Factor de Unión al Sitio Principal/genética , Humanos , Concentración 50 Inhibidora , Integrina beta3/metabolismo , Megacariocitos/citología , Megacariocitos/metabolismo , Mitocondrias/metabolismo , Hojas de la Planta/química , Plantas Medicinales/química , Glicoproteína IIb de Membrana Plaquetaria/metabolismo , Proteína 3 Supresora de la Señalización de Citocinas/genética , Proteína 3 Supresora de la Señalización de Citocinas/metabolismo , Trombopoyesis/efectos de los fármacosRESUMEN
Lusutrombopag (S-888711), an oral small-molecule thrombopoietin receptor (TPOR) agonist, has gained first approval as a drug to treat thrombocytopenia of chronic liver disease in patients undergoing elective invasive procedures in Japan. Preclinical studies were performed to evaluate its efficacy against megakaryopoiesis and thrombopoiesis. To investigate the proliferative activity and efficacy of megakaryocytic colony formation via human TPOR, lusutrombopag was applied to cultured human c-Mpl-expressing Ba/F3 (Ba/F3-hMpl) cells and human bone marrow-derived CD34-positive cells, respectively. Lusutrombopag caused a robust increase in Ba/F3-hMpl cells by activating pathways in a manner similar to that of thrombopoietin and induced colony-forming units-megakaryocyte and polyploid megakaryocytes in human CD34-positive cells. Because lusutrombopag has high species specificity for human TPOR, there was no suitable experimental animal model for drug evaluation, except for immunodeficient mouse-based xenograft models. Therefore, a novel genetically modified knock-in mouse, TPOR-Ki/Shi, was developed by replacing mouse Mpl with human-mouse chimera Mpl. In TPOR-Ki/Shi mice, lusutrombopag significantly increased circulating platelets in a dose-dependent manner during 21-day repeated oral administration. Histopathological study of the TPOR-Ki/Shi mice on day 22 also revealed a significant increase in megakaryocytes in the bone marrow. These results indicate that lusutrombopag acts on human TPOR to upregulate differentiation and proliferation of megakaryocytic cells, leading to platelet production.
Asunto(s)
Proliferación Celular/efectos de los fármacos , Cinamatos/farmacología , Megacariocitos/metabolismo , Modelos Biológicos , Receptores de Trombopoyetina/agonistas , Tiazoles/farmacología , Animales , Plaquetas/citología , Plaquetas/metabolismo , Línea Celular , Evaluación Preclínica de Medicamentos , Técnicas de Sustitución del Gen , Humanos , Megacariocitos/citología , Ratones , Ratones Transgénicos , Receptores de Trombopoyetina/genética , Receptores de Trombopoyetina/metabolismoRESUMEN
Immune thrombocytopenia (ITP) is an immune-mediated acquired bleeding disorder characterized by abnormally low platelet counts. We reported here the ability of low-level light treatment (LLLT) to alleviate ITP in mice. The treatment is based on noninvasive whole body illumination 30 min a day for a few consecutive days by near infrared light (830 nm) transmitted by an array of light-emitting diodes (LEDs). LLLT significantly lifted the nadir of platelet counts and restored tail bleeding time when applied to two passive ITP models induced by anti-CD41 antibody. The anti-platelet antibody hindered megakaryocyte differentiation from the progenitors, impaired proplatelet and platelet formation, and induced apoptosis of platelets. These adverse effects of anti-CD41 antibody were all mitigated by LLLT to varying degrees, owing to its ability to enhance mitochondrial biogenesis and activity in megakaryocytes and preserve mitochondrial functions in platelets in the presence of the antibody. The observations argue not only for contribution of mitochondrial stress to the pathology of ITP, but also clinical potentials of LLLT as a safe, simple, and cost-effective modality of ITP.
Asunto(s)
Diferenciación Celular/efectos de la radiación , Terapia por Luz de Baja Intensidad/métodos , Megacariocitos/efectos de la radiación , Trombocitopenia/radioterapia , Animales , Apoptosis/inmunología , Apoptosis/efectos de la radiación , Diferenciación Celular/inmunología , Megacariocitos/citología , Megacariocitos/inmunología , Ratones Endogámicos C57BL , Recuento de Plaquetas , Trombocitopenia/inmunología , Trombopoyesis/inmunología , Trombopoyesis/efectos de la radiaciónRESUMEN
Constitutional thrombocytopenias result from platelet production abnormalities of hereditary origin. Long misdiagnosed and poorly studied, knowledge about these rare diseases has increased considerably over the last twenty years due to improved technology for the identification of mutations, as well as an improvement in obtaining megakaryocyte culture from patient hematopoietic stem cells. Simultaneously, the manipulation of mouse genes (transgenesis, total or conditional inactivation, introduction of point mutations, random chemical mutagenesis) have helped to generate disease models that have contributed greatly to deciphering patient clinical and laboratory features. Most of the thrombocytopenias for which the mutated genes have been identified now have a murine model counterpart. This review focuses on the contribution that these mouse models have brought to the understanding of hereditary thrombocytopenias with respect to what was known in humans. Animal models have either i) provided novel information on the molecular and cellular pathways that were missing from the patient studies; ii) improved our understanding of the mechanisms of thrombocytopoiesis; iii) been instrumental in structure-function studies of the mutated gene products; and iv) been an invaluable tool as preclinical models to test new drugs or develop gene therapies. At present, the genetic determinants of thrombocytopenia remain unknown in almost half of all cases. Currently available high-speed sequencing techniques will identify new candidate genes, which will in turn allow the generation of murine models to confirm and further study the abnormal phenotype. In a complementary manner, programs of random mutagenesis in mice should also identify new candidate genes involved in thrombocytopenia.
Asunto(s)
Trombocitopenia/etiología , Trombocitopenia/metabolismo , Animales , Autoantígenos/metabolismo , Síndrome de Bernard-Soulier/etiología , Síndrome de Bernard-Soulier/metabolismo , Plaquetas/metabolismo , Diferenciación Celular/genética , Citoesqueleto/metabolismo , Modelos Animales de Enfermedad , Regulación de la Expresión Génica , Humanos , Yoduro Peroxidasa/metabolismo , Proteínas de Unión a Hierro/metabolismo , Megacariocitos/citología , Megacariocitos/metabolismo , Ratones , Receptores de Trombopoyetina/metabolismo , Transducción de Señal , Trombocitopenia/diagnóstico , Trombopoyesis , Factores de Transcripción/metabolismo , Síndrome de Wiskott-Aldrich/etiología , Síndrome de Wiskott-Aldrich/metabolismoRESUMEN
OBJECTIVE: To investigate the effects of panaxadiol saponins component (PDS-C) isolated from total saponins of panax ginseng on proliferation, differentiation and corresponding gene expression profile of megakaryocytes. METHODS: Bone marrow culture of colony forming assay of megakaryocytic progenitor cells (CFU-MK) was observed for the promoting proliferation mediated by PDS-C, and differentiation of megakaryocytic blasts caused by PDS-C was analyzed with flow cytometry in CHRF-288 and Meg-01 cells, as well as proliferation, differentiation-related genes expression profile and protein expression levels were detected by human gene expression microarray and western blot. RESULTS: In response to PDS-C 10, 20 and 50 mg/L, CFU-MK from 10 human bone marrow samples was increased by 28.9%±2.7%, 41.0%±3.2% and 40.5%±2.6% over untreated control, respectively (P <0.01, each). Flow cytometry analysis showed that PDS-C treated CHRF-288 cells and Meg-01 cells significantly increased in CD42b, CD41, TSP and CD36 positive ratio, respectively. PDS-C induced 29 genes up-regulated more than two-fold commonly in both cells detected by human expression microarray representing 4000 known genes. The protein expression levels of ZNF91, c-Fos, BTF3a, GATA-1, RGS2, NDRG2 and RUNX1 were increased with western blot in correspond to microarray results. CONCLUSION: PDS-C as an effective component for hematopoiesis, play the role to enhance proliferation and differentiation of megakaryocytes, also up-regulated expression of proliferation, differentiation-related genes and proteins in vitro.
Asunto(s)
Diferenciación Celular/efectos de los fármacos , Medicamentos Herbarios Chinos/farmacología , Perfilación de la Expresión Génica , Ginsenósidos/farmacología , Megacariocitos/citología , Megacariocitos/metabolismo , Patentes como Asunto , Saponinas/farmacología , Western Blotting , Células de la Médula Ósea/citología , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Ensayo de Unidades Formadoras de Colonias , Citometría de Flujo , Humanos , Megacariocitos/efectos de los fármacos , Células Madre/citología , Células Madre/efectos de los fármacos , Factores de Transcripción/metabolismo , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/genéticaRESUMEN
Nicotinamide adenine dinucleotide (NAD) is an essential co-factor in glycolysis and is a key molecule involved in maintaining cellular energy metabolism. Nicotinamide phosphoribosyltransferase (NAMPT) catalyzes the rate-limiting step of an important salvage pathway in which nicotinamide is recycled into NAD. NAMPT is up-regulated in many types of cancer and NAMPT inhibitors (NAMPTi) have potential therapeutic benefit in cancer by impairing tumor metabolism. Clinical trials with NAMPTi APO-866 and GMX-1778, however, failed to reach projected efficacious exposures due to dose-limiting thrombocytopenia. We evaluated preclinical models for thrombocytopenia that could be used in candidate drug selection and risk mitigation strategies for NAMPTi-related toxicity. Rats treated with a suite of structurally diverse and potent NAMPTi at maximum tolerated doses had decreased reticulocyte and lymphocyte counts, but no thrombocytopenia. We therefore evaluated and qualified a human colony forming unit-megakaryocyte (CFU-MK) as in vitro predictive model of NAMPTi-induced MK toxicity and thrombocytopenia. We further demonstrate that the MK toxicity is on-target based on the evidence that nicotinic acid (NA), which is converted to NAD via a NAMPT-independent pathway, can mitigate NAMPTi toxicity to human CFU-MK in vitro and was also protective for the hematotoxicity in rats in vivo. Finally, assessment of CFU-MK and human platelet bioenergetics and function show that NAMPTi was toxic to MK and not platelets, which is consistent with the clinically observed time-course of thrombocytopenia.
Asunto(s)
Antineoplásicos/efectos adversos , Inhibidores Enzimáticos/efectos adversos , Hematopoyesis/efectos de los fármacos , Megacariocitos/efectos de los fármacos , Niacina/metabolismo , Nicotinamida Fosforribosiltransferasa/antagonistas & inhibidores , Trombocitopenia/inducido químicamente , Animales , Antineoplásicos/química , Plaquetas/efectos de los fármacos , Plaquetas/metabolismo , Células Cultivadas , Ensayo de Unidades Formadoras de Colonias , Suplementos Dietéticos , Evaluación Preclínica de Medicamentos , Inhibidores Enzimáticos/química , Interacciones Alimento-Droga , Humanos , Leucocitos Mononucleares/citología , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/metabolismo , Leucocitos Mononucleares/patología , Macaca fascicularis , Masculino , Megacariocitos/citología , Megacariocitos/metabolismo , Megacariocitos/patología , Ratones , Estructura Molecular , Niacina/uso terapéutico , Nicotinamida Fosforribosiltransferasa/genética , Nicotinamida Fosforribosiltransferasa/metabolismo , Pentosiltransferasa/genética , Pentosiltransferasa/metabolismo , Ratas Sprague-Dawley , Trombocitopenia/metabolismo , Trombocitopenia/prevención & controlRESUMEN
Thrombocytopenia (TTP) is a blood disease common to canines and human beings. Currently, there is no valid therapy for this disease except blood transfusion. In this study, we report the generation of canine induced pluripotent stem cells (ciPSCs) from canine embryonic fibroblasts, and a novel protocol for creating mature megakaryocytes (MKs) and functional platelets from ciPSCs. The ciPSCs were generated using lentiviral vectors, and differentiated into MKs and platelets on OP9 stromal cells supplemented with growth factors. Our ciPSCs presented in a tightly domed shape and showed expression of a critical pluripotency marker, REX1, and normal karyotype. Additionally, ciPSCs differentiated into cells derived from three germ layers via the formation of an embryoid body. The MKs derived from ciPSCs had hyperploidy and transformed into proplatelets. The proplatelets released platelets early on that expressed specific MK and platelet marker CD41/61. Interestingly, these platelets, when activated with adenosine diphosphate or thrombin, bind to fibrinogen. Moreover, electron microscopy showed that the platelets had the same ultrastructure as peripheral platelets. Thus, we have demonstrated for the first time the generation of ciPSCs that are capable of differentiating into MKs and release functional platelets in vitro. Our system for differentiating ciPSCs into MKs and platelets promises a critical therapy for canine TTP and appears to be extensible in principle to resolve human TTP.
Asunto(s)
Plaquetas/metabolismo , Fibroblastos/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Megacariocitos/metabolismo , Adenosina Difosfato/farmacología , Animales , Plaquetas/citología , Plaquetas/efectos de los fármacos , Diferenciación Celular , Células Cultivadas , Perros , Embrión de Mamíferos , Cuerpos Embrioides , Fibrinógeno/química , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Expresión Génica , Vectores Genéticos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Péptidos y Proteínas de Señalización Intercelular/farmacología , Factores de Transcripción de Tipo Kruppel/genética , Factores de Transcripción de Tipo Kruppel/metabolismo , Lentivirus , Megacariocitos/citología , Megacariocitos/efectos de los fármacos , Glicoproteína IIb de Membrana Plaquetaria/genética , Glicoproteína IIb de Membrana Plaquetaria/metabolismo , Unión Proteica , Trombina/farmacologíaRESUMEN
Hematopoietic stem and progenitor cells (HSPCs) have been cultured using a wide variety of cytokines to promote differentiation into megakaryocytic cells (Mks), the precursors to platelets. Greater Mk DNA content, or ploidy, has been correlated with increased platelet release. Gradients of pH, pO2, and signaling factors regulate megakaryopoiesis in the bone marrow niche. In this study, we demonstrate that a 3-phase culture process with increasing pH and pO2 and different cytokine cocktails greatly increases megakaryocyte production. CD34(+) HSPCs were first cultured at 5% O2 and pH 7.2 with a cytokine cocktail previously shown to promote Mk progenitor production. At day 5, cells were shifted to 20% O2 and pH 7.4 and maintained in 1 of 17 cytokine cocktails identified using a 2(4) factorial design of experiments method to evaluate the effects of interleukin (IL)-3, IL-6, IL-9, and high- or low-dose stem cell factor (SCF), in conjunction with thrombopoietin (Tpo) and IL-11, on expansion of mature Mks from progenitors. The combination of Tpo, high-dose SCF, IL-3, IL-9, and IL-11 best promoted Mk expansion. IL-3 greatly increased total cell fold expansion, but this was partially offset by lower Mk purity. IL-9 promoted CD41 and CD42b expression. High-dose (100 ng/mL) SCF increased Mk production and ploidy. Different commercial media and IL-3 sources substantially impacted differentiation, and X-VIVO 10 serum-free media best supported mature Mk expansion. Shifting from pH 7.4 to pH 7.6 at day 7 increased Mk production by 30%. Treatment with nicotinamide at day 7 or day 8 more than doubled the fraction of high-ploidy (>4N) Mks. Ultimately, the 3-phase culture system gave rise to 44.5±8.1 Mks and 8.5±3.1 high-ploidy Mks per input HSPC. Further optimization was required to improve platelet production. Using Iscove's modified Dulbecco's medium (IMDM)+20% BSA, insulin and transferin (BIT) 9500 Serum Substitute greatly improved the frequency and quality of Mk proplatelet extensions without affecting Mk expansion, commitment, or polyploidization in the 3-phase process. Mks cultured in IMDM+20% BIT 9500 gave rise to platelets with functional activity similar to that of fresh platelets from normal donors, as evidenced by basal tubulin distribution and the expression of surface markers and spreading in response to platelet agonists.
Asunto(s)
Plaquetas/citología , Técnicas de Cultivo de Célula/métodos , Megacariocitos/citología , Ploidias , Biomarcadores/metabolismo , Plaquetas/efectos de los fármacos , Plaquetas/metabolismo , Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Forma de la Célula/efectos de los fármacos , Células Cultivadas , Medio de Cultivo Libre de Suero , Citocinas/farmacología , Humanos , Concentración de Iones de Hidrógeno , Megacariocitos/efectos de los fármacos , Megacariocitos/metabolismo , Niacinamida/farmacología , Selectina-P/metabolismo , Activación Plaquetaria/efectos de los fármacos , Tetraspanina 30/metabolismoRESUMEN
Hematopoietic processes, especially megakaryocytopoiesis and thrombopoiesis, are highly sensitive to extracellular oxidative stresses such as ionizing radiation and chemotherapeutic agents. This study examined the terminal maturation of megakaryocytes and platelet production in hematopoietic stem/progenitor cells (HSPCs) exposed to ionizing radiation. Highly purified CD34(+) cells derived from human placental/umbilical cord blood were exposed to X rays (2 Gy, 150 kVp, 20 mA; 0.5-mm aluminum and 0.3-mm copper filters) at a dose rate of approximately 1 Gy/min and then cultured in a serum-free medium supplemented with thrombopoietin and interleukin-3. The number of cells generated from X-irradiated CD34(+) cells decreased with the time in culture. However, the fraction of CD34(+)Tie-2(+) and CD41(+)Tie-2(+) cells among the total cells generated from X-irradiated cells increased significantly in comparison to nonirradiated controls on day 7. In addition, the CD42a(+) particles, which appeared to be platelets, generated from the X-irradiated HSPCs appeared to be normal. Quantitative real-time reverse transcriptase-polymerase chain reaction analysis of the expression of various genes in cells harvested from the cultures showed that the early hematopoiesis-related genes FLI1, HOXB4 and Tie-2, the cytokine receptor genes KIT and IL3RA, and the oxidative stress-related genes HO1 and NQO1 were upregulated on day 7. These results suggest that normal terminal maturation of megakaryocytes and platelet production occur in residual HSPCs after exposure to ionizing radiation despite the adverse effect of radiation on proliferation and differentiation of HSPCs. Ionizing radiation may have the potential to promote both megakaryocytopoiesis and thrombopoiesis.
Asunto(s)
Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/efectos de la radiación , Trombopoyesis/efectos de la radiación , Antígenos CD34/metabolismo , Plaquetas/citología , Plaquetas/efectos de la radiación , Femenino , Sangre Fetal/citología , Regulación de la Expresión Génica/efectos de la radiación , Células Madre Hematopoyéticas/metabolismo , Humanos , Interleucina-3/metabolismo , Megacariocitos/citología , Megacariocitos/efectos de la radiación , Placenta/citología , Placenta/efectos de la radiación , Glicoproteína IIb de Membrana Plaquetaria/metabolismo , Embarazo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Dosis de Radiación , Trombopoyetina/metabolismoRESUMEN
BACKGROUND AIMS: Ex vivo generation of megakaryocytes (MK) from hematopoietic stem cells (HSC) is important for both basic research, to understand the mechanism of platelet biogenesis, and clinical infusions, for rapid platelet recovery in thrombocytopenic patients. We investigated the role of two nutraceuticals, docosahexanoic acid (DHA) and arachidonic acid (AA), in the in vitro generation of MK. METHODS: Umbilical cord blood (UCB)-derived CD34+cells were cultured with stem cell factor (SCF) and thrombopoietin (TPO) in the presence (test) or absence (control) of the two additives. On day 10, MK and platelets generated were quantitated by morphologic, phenotypic and functional assays. RESULTS: The cell yield of MK and platelet numbers were significantly higher in test compared with control cells. Phenotypic analyzes and gene expression profiles confirmed these findings. Functional properties, such as colony-forming unit (CFU)-MK formation, chemotaxis and platelet activation, were found to be enhanced in cells cultured with nutraceuticals. The engraftment potential of ex vivo-expanded cells was studied in NOD/SCID mice. Mice that received MK cultured in the presence of DHA/AA engrafted better. There was a reduction in apoptosis and total reactive oxygen species (ROS) levels in the CD41(+) compartment of the test compared with control sets. The data suggest that these compounds probably exert their beneficial effect by modulating apoptotic and redox pathways. CONCLUSIONS: Use of nutraceuticals like DHA and AA may prove to be a useful strategy for efficient generation of MK and platelets from cord blood cells, for future use in clinics and basic research.
Asunto(s)
Antígenos CD34/metabolismo , Ácido Araquidónico/farmacología , Medios de Cultivo/química , Citocinas/farmacología , Ácidos Docosahexaenoicos/farmacología , Sangre Fetal/citología , Megacariocitos/citología , Animales , Apoptosis/efectos de los fármacos , Plaquetas/citología , Plaquetas/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Quimiotaxis/efectos de los fármacos , Ensayo de Unidades Formadoras de Colonias , Suplementos Dietéticos , Humanos , Megacariocitos/trasplante , Ratones , Ratones SCID , Fenotipo , Activación Plaquetaria/efectos de los fármacos , Ploidias , Especies Reactivas de Oxígeno/metabolismo , Factores de Transcripción/metabolismoRESUMEN
BACKGROUND: In patients transplanted with cord blood (CB), prolonged thrombocytopenia is a major complication. However, this could be alleviated by supplementing the CB graft with ex vivo-expanded megakaryocytic progenitors (CFU-Meg), provided that the homing properties of these cells are not affected negatively by expansion. METHODS AND RESULTS: We assessed the in vitro homing potential of CFU-Meg progenitors expanded from CB and showed that the combination of thrombopoietin (TPO) with interleukin-3 (IL-3) used for expansion not only results in optimal proliferation of CFU-Meg but also protects these cells from apoptosis. Moreover, we found that ex vivo-expanded CFU-Meg maintained expression of the CXCR4 receptor throughout a 9-day culture and were chemoattracted towards a stromal cell-derived factor-1 (SDF-1) gradient. They also expressed matrix metalloproteinase-9 (MMP-9) and membrane-type (MT) 1-MMP, and transmigrated across the reconstituted basement membrane Matrigel. Finally, we observed that SDF-1 up-regulated the expression of both MMP-9 and MT1-MMP in CB CD34(+) cells and ex vivo-expanded CFU-Meg. DISCUSSION: We suggest that CB-expanded CFU-Meg, in particular those from day 3 of expansion, when their proliferation and in vitro homing potential are maximal, could be employed to supplement CB grafts and speed up platelet recovery in transplant recipients.
Asunto(s)
Ensayo de Unidades Formadoras de Colonias , Sangre Fetal/citología , Sangre Fetal/enzimología , Metaloproteinasa 14 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Megacariocitos/citología , Células Madre/citología , Antígenos CD34/metabolismo , Catequina/análogos & derivados , Catequina/farmacología , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Quimiocina CXCL12/metabolismo , Factores Quimiotácticos/farmacología , Quimiotaxis/efectos de los fármacos , Colágeno/metabolismo , Combinación de Medicamentos , Sangre Fetal/efectos de los fármacos , Humanos , Interleucina-3/farmacología , Cinética , Laminina/metabolismo , Metaloproteinasa 14 de la Matriz/genética , Metaloproteinasa 9 de la Matriz/genética , Megacariocitos/efectos de los fármacos , Megacariocitos/enzimología , Glicoproteína IIb de Membrana Plaquetaria/metabolismo , Proteoglicanos/metabolismo , Receptores CXCR4/metabolismo , Células Madre/efectos de los fármacos , Trombopoyetina/farmacología , Regulación hacia Arriba/efectos de los fármacosRESUMEN
The in vitro biosynthesis of metallothionein (MT) was investigated in thrombocyte precursors (megakaryocytes) isolated from human cord blood. Biosynthesis and induction of MT in magnetic cell sorting-separated CD61(+) megakaryocytes was confirmed by immunohistochemical staining using monoclonal mouse anti-MT. The presence of MT was detected both in the nuclear and in the cytoplasmic area. Using RT-PCR, in vitro upregulation/induction of total MT transcripts was observed in CD61(+) cells at 48 h post-treatment with 100 micromol/L of zinc supplement. Seven isoform-specific mRNAs namely, MT-1A, MT-1B, MT-1E, MT-1G, MT-1H, MT-1X, and MT-2A were detected in the similar cell populations left untreated with zinc.
Asunto(s)
Plaquetas/citología , Plaquetas/metabolismo , Integrina beta3/metabolismo , Megacariocitos/metabolismo , Metalotioneína/metabolismo , Células Madre/metabolismo , Plaquetas/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Inmunohistoquímica , Recién Nacido , Megacariocitos/citología , Megacariocitos/efectos de los fármacos , Metalotioneína/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Transporte de Proteínas/efectos de los fármacos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Células Madre/efectos de los fármacos , Zinc/farmacologíaRESUMEN
Gfi1b and Gfi1 are 37- and 55-kDa transcriptional repressors that share common features such as a 20-amino acid (aa) N-terminal SNAG domain, a nonconserved intermediary domain, and 6 highly conserved C-terminal zinc fingers. Both gene loci are under autoregulatory and cross-regulatory feedback control. We have generated a reporter mouse strain by inserting the cDNA for green fluorescent protein (GFP) into the Gfi1b gene locus which allowed us to follow Gfi1b expression during hematopoiesis and lymphopoiesis by measuring green fluorescence. We found highly dynamic expression patterns of Gfi1b in erythroid cells, megakaryocytes, and their progenitor cells (MEPS) where Gfi1 is not detected. Vice versa, Gfi1b could not be found in granulocytes, activated macrophages, or their granulomonocytic precursors (GMPs) or in mature naive or activated lymphocytes where Gfi1 is expressed, suggesting a complementary regulation of both loci during hematopoiesis. However, Gfi1b was found to be up-regulated in early stages of B-cell and in a subset of early T-cell development, where Gfi1 is also present, suggesting that cross-regulation of both loci exists but is cell-type specific.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Expresión Génica , Proteínas Fluorescentes Verdes/metabolismo , Hematopoyesis , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Represoras/metabolismo , Factores de Transcripción/metabolismo , Envejecimiento/fisiología , Animales , Biomarcadores , Linaje de la Célula , Células Cultivadas , Embrión de Mamíferos/citología , Embrión de Mamíferos/embriología , Embrión de Mamíferos/metabolismo , Células Eritroides/citología , Células Eritroides/metabolismo , Proteínas Fluorescentes Verdes/genética , Megacariocitos/citología , Megacariocitos/metabolismo , Ratones , Ratones Transgénicos , Células Mieloides/metabolismo , Proteínas Proto-Oncogénicas/genética , Proteínas Represoras/genética , Células Madre/metabolismo , Subgrupos de Linfocitos T/citología , Subgrupos de Linfocitos T/metabolismo , Linfocitos T/citología , Linfocitos T/metabolismoRESUMEN
It has previously been shown that Dendrostellera lessertii (Thymelaeaceae) has strong anticancer activity. In this study, the antileukemic activity of another new compound from the same plant extract is reported. Promyelocytic (NB4 and HL-60) and erythroleukemia (K562) cells were cultured in the presence of various concentrations of the new compound (0.5-3.0 mug/ml) for 3 d. The cell numbers were then determined by trypan blue exclusion test. The new compound inhibited growth and proliferation of NB4, HL-60 and K562 with IC50 values of 1.5, 2.0 and 2.5 mg/ml, respectively. We also found that the new compound inhibited cell proliferation in a dose- and time-dependent manner. At low concentrations and after 48 h of treatment, approximately 50%-70% of NB4 and HL-60 cells were differentiated to monocyte/macrophage lineage and approximately 30%-40% of the treated K562 cells were differentiated in the megakaryocytic lineage, as evidenced by morphological changes and nitro blue tetrazolium reduction assays. Results of Hoechst 33258 staining also indicated that the new compound induced NB4 and HL-60 cell apoptosis at their respective IC50 values after 72 h of treatment. Based on the present data, the new compound seems a good candidate for further evaluation as an effective chemotherapeutic agent acting through induction of differentiation and apoptosis.
Asunto(s)
Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Leucemia/patología , Extractos Vegetales/farmacología , Thymelaeaceae/química , Línea Celular Tumoral , Linaje de la Célula , Supervivencia Celular/efectos de los fármacos , Fragmentación del ADN , Relación Dosis-Respuesta a Droga , Células HL-60 , Humanos , Macrófagos/citología , Megacariocitos/citología , Fagocitosis/efectos de los fármacosRESUMEN
Epigallocatechin-3-gallate (EGCg) has been widely recognized as a powerful antioxidant and free radical scavenger. The effects of EGCg on the proliferation and differentiation of X-irradiated megakaryocytic progenitor cells (colony-forming unit-megakaryocyte, CFU-Meg) using CD34+ cells prepared from human placental and umbilical cord blood have been shown. In the absence of exogenous thrombopoietin (TPO), no colonies are observed in cultures containing or lacking EGCg (1 nM-100 microM). In the presence of TPO, in contrast, EGCg significantly promotes CFU-Meg-derived colony formations within the 10-100-nM range. A 1.5-fold increase in the total number of CFU-Meg has been counted compared with the control. These favorable effects of EGCg are also observed in the culture of CD34+ cells before and after X irradiation with 2 Gy. Moreover, in order to investigate the function of EGCg promoting megakaryocytopoiesis and thrombopoiesis in ex vivo cultures, both non-irradiated and X-irradiated CD34+ cells are grown in liquid cultures supplemented with TPO. In both cultures, EGCg increases the total number of cells and megakaryocytes. It has been suggested that the favorable effects of EGCg reduce the risk factor from radiation damage in megakaryocytopoiesis.