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1.
Rinsho Ketsueki ; 60(9): 1056-1062, 2019.
Artigo em Japonês | MEDLINE | ID: mdl-31597827

RESUMO

The hematopoietic stem cells, defined as blood stem cells with self-replication ability and multipotency, are key to successful hematopoietic stem cell transplantation. With the history of transplantation in the past 60 years and advances in stem cell technologies, our understanding of the hematopoietic system has deepened. However, the molecular mechanisms of self-renewal and pluripotency, which are the essence of the hematopoietic stem cells, remain poorly understood. One reason is that the identification/purification methods of the hematopoietic stem cells, particularly the long-term hematopoietic stem cells capable of lifelong self-renewal, is technically difficult owing to their scarcity in the bone marrow and has not been established to this date. Considering that a long-lasting blood production after hematopoietic stem cell transplantation is crucial, it is essential to understand the biology of the long-term hematopoietic stem cells not only scientifically but also clinically. This review describes the scientific and clinical significance of the long-term hematopoietic stem cells by showing the results of the latest researches in the introduction of hematopoietic stem cell identification/purification history.


Assuntos
Separação Celular , Células-Tronco Hematopoéticas/citologia , Medula Óssea , Células da Medula Óssea/citologia , Transplante de Células-Tronco Hematopoéticas , Humanos
2.
Rinsho Ketsueki ; 60(9): 1075-1083, 2019.
Artigo em Japonês | MEDLINE | ID: mdl-31597830

RESUMO

The mechanism underlying production of various types of blood cells from hematopoietic stem and progenitor cells has been a central theme in hematology. Conventionally, hematopoietic cell populations are analyzed by cell surface markers to judge cell types and differentiation stages, and by transplantation assays to assess differentiation potential. Recently, however, next-generation sequencing technology has enabled single-cell transcriptome and epigenome analyses and cell barcoding-based lineage tracing during unperturbed hematopoiesis. These innovative assays revealed that each cell population is extensively heterogenous. Many cells within hematopoietic stem cell populations may not be multipotent, and conversely, hematopoietic progenitor cells often display self-renewal capacity. Moreover, cells tend to make their lineage choice much earlier than previously thought. Altogether, these results challenge the current hierarchical differentiation models and propose new continuous models. Single-cell analyses are expected to greatly contribute to our understanding of normal and abnormal hematopoiesis and to the development of new therapies for blood disorders.


Assuntos
Hematopoese , Células-Tronco Hematopoéticas/citologia , Análise de Célula Única , Diferenciação Celular , Linhagem da Célula , Epigenômica , Humanos , Transcriptoma
3.
Mol Biol (Mosk) ; 53(5): 711-724, 2019.
Artigo em Russo | MEDLINE | ID: mdl-31661473

RESUMO

Blood is extremely important for a multicellular organism: it connects all organs and tissues, supplies them with nutrients and oxygen, removes carbon dioxide and metabolic products, maintains homeostasis, and provides protection against infections. That is why studies on blood have always drawn a great deal of attention. In ancient times, it was believed that the soul was in the blood and that it sometimes "sank into the stomach." Initially, the study of blood was limited to morphological methods, to which physiological and cellular research were added in the twentieth century. With their help, researchers established that mature blood cells are formed from a rare population of hematopoietic stem cells (HSCs), which are located in the bone marrow. The development of molecular biology methods and their combination with classical physiological ones allowed a breakthrough in understanding the structure of the hematopoietic system, which changed our understanding not only of hematopoiesis but also about the nature of adult stem cells. This review describes the molecular assays used in experimental hematology, and how their application has gradually been expanding our knowledge of blood formation and continues to provide new information about it.


Assuntos
Hematopoese , Sistema Hematopoético/citologia , Sistema Hematopoético/fisiologia , Biologia Molecular/métodos , Células-Tronco Adultas/citologia , Medula Óssea , Células-Tronco Hematopoéticas/citologia , Humanos
4.
Adv Exp Med Biol ; 1143: 59-74, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31338815

RESUMO

One of the bottlenecks of the treatments for malignant hematopoietic disorders is the unavailability of sufficient amount of hematopoietic stem cells (HSCs). HSCs are considered to be originated from the aorta-gonad-mesonephros and gradually migrates into fetal liver and resides in a unique microenvironment/niche of bone marrow. Although many intrinsic and extrinsic factors (niche components) are reported to be involved in the origination, maturation, migration, and localization of HSCs at different developmental stages, the detailed molecular mechanisms still remain largely unknown. Previous studies have shown that intrinsic metabolic networks may be critical for the cell fate determinations of HSCs. For example, HSCs mainly utilize glycolysis as the main energy sources; oxidative phosphorylation is required for the homeostasis of HSCs; lipid or amino acid metabolisms may also sustain HSC stemness. Mechanistically, lots of regulatory pathways, such as MEIS1/HIF1A and PI3K/AKT/mTOR signaling, are found to fine-tune the different nutrient metabolisms and cell fate commitments of HSCs. However, more efforts are required for the optimization and establishment of precise metabolic techniques specific for the HSCs with relatively rare cell frequency and understanding of the basic metabolic properties and their underlying regulatory mechanisms of different nutrients (such as glucose) during the different developmental stages of HSCs.


Assuntos
Diferenciação Celular , Células-Tronco Hematopoéticas , Transdução de Sinais , Medula Óssea/metabolismo , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Humanos
5.
Life Sci ; 232: 116598, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31247209

RESUMO

Hematopoietic stem cells (HSCs) are a rare cell population in adult bone marrow, mobilized peripheral blood, and umbilical cord blood possessing self-renewal and differentiation capability into a full spectrum of blood cells. Bone marrow HSC transplantation has been considered as an ideal option for certain disorders treatment including hematologic diseases, leukemia, immunodeficiency, bone marrow failure syndrome, genetic defects such as thalassemia, sickle cell anemia, autoimmune disease, and certain solid cancers. Ex vivo proliferation of these cells prior to transplantation has been proposed as a potential solution against limited number of stem cells. In such culture process, MSCs have also been shown to exhibit high capacity for secretion of soluble mediators contributing to the principle biological and therapeutic activities of HSCs. In addition, endothelial cells have been introduced to bridge the blood and sub tissues in the bone marrow, as well as, HSCs regeneration induction and survival. Cell culture in the laboratory environment requires cell growth strict control to protect against contamination, symmetrical cell division and optimal conditions for maximum yield. In this regard, microfluidic systems provide culture and analysis capabilities in micro volume scales. Moreover, two-dimensional cultures cannot fully demonstrate extracellular matrix found in different tissues and organs as an abstract representation of three dimensional cell structure. Microfluidic systems can also strongly describe the effects of physical factors such as temperature and pressure on cell behavior.


Assuntos
Transplante de Células-Tronco Hematopoéticas/métodos , Células-Tronco Hematopoéticas/citologia , Animais , Células da Medula Óssea/citologia , Técnicas de Cultura de Células/métodos , Técnicas de Cocultura , Células Endoteliais/citologia , Sangue Fetal/citologia , Humanos , Células-Tronco Mesenquimais/citologia
6.
Nat Immunol ; 20(7): 802-811, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31213716

RESUMO

Recent advances have highlighted the ability of hematopoietic stem and progenitor cells in the bone marrow to sense peripheral inflammation or infection and adapt through increased proliferation and skewing toward the myeloid lineage. Such adaptations can meet the increased demand for innate immune cells and can be beneficial in response to infection or myeloablation. However, the inflammation-induced adaptation of hematopoietic and myeloid progenitor cells toward enhanced myelopoiesis might also perpetuate inflammation in chronic inflammatory or cardio-metabolic diseases by generating a feed-forward loop between inflammation-adapted hematopoietic progenitor cells and the inflammatory disorder. Sustained adaptive responses of progenitor cells in the bone marrow can also contribute to trained immunity, a non-specific memory of earlier encounters that in turn facilitates the heightened response of these cells, as well as that of their progeny, to future challenges. Here we discuss the mechanisms that govern the adaptation of hematopoietic progenitor cells to inflammation and its sequelae in the pathogenesis of human disease.


Assuntos
Adaptação Biológica , Células-Tronco Hematopoéticas/fisiologia , Inflamação/etiologia , Inflamação/metabolismo , Animais , Citocinas/metabolismo , Células-Tronco Hematopoéticas/citologia , Humanos , Imunidade , Imunomodulação , Inflamação/patologia , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Interferons/metabolismo , Mielopoese , Transdução de Sinais , Receptores Toll-Like/metabolismo
7.
Autoimmun Rev ; 18(8): 751-760, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31181324

RESUMO

Neutrophils derive from hematopoietic stem cells (HSCs) with systemic inflammation driving their activation and differentiation to myeloid progenitors to ensure enhanced myelopoiesis. Epigenetic reprograming and re-education of these HSCs produces neutrophils primed towards elimination of pathogens and increased inflammatory response. Neutrophils -an important component of acute inflammation- are not present in chronic inflammatory tissues leading to the false assumption that they may not be as important for the latter. Activated neutrophils may release Neutrophil Extracellular Traps (NETs) during a distinct form of cell death, named NETosis; NETs are rich in bioactive molecules that promote thrombosis (including atherothrombosis), inflammation and fibrosis. Thus, although neutrophils may not be present in chronic inflammatory lesions, their remnants may amplify the inflammatory response beyond their short life-span in the tissues. Herein, we review current evidence supporting a role of neutrophils and NETosis in tissue injury and dysfunction in systemic autoimmunity using as disease paradigms Systemic Lupus Erythematosus (SLE) and the ANCA-associated vasculitides (AAV). We also discuss the mechanisms involved and their potential as targets for novel therapy and drug repositioning.


Assuntos
Vasculite Associada a Anticorpo Anticitoplasma de Neutrófilos/imunologia , Lúpus Eritematoso Sistêmico/imunologia , Neutrófilos/imunologia , Animais , Vasculite Associada a Anticorpo Anticitoplasma de Neutrófilos/patologia , Vasculite Associada a Anticorpo Anticitoplasma de Neutrófilos/terapia , Morte Celular , Diferenciação Celular , Armadilhas Extracelulares/imunologia , Fibrose , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/imunologia , Humanos , Inflamação/imunologia , Inflamação/patologia , Inflamação/terapia , Lúpus Eritematoso Sistêmico/patologia , Lúpus Eritematoso Sistêmico/terapia , Mielopoese , Neutrófilos/patologia
8.
Rinsho Ketsueki ; 60(5): 453-458, 2019.
Artigo em Japonês | MEDLINE | ID: mdl-31168013

RESUMO

Differentiation of hematopoietic stem/progenitor cells should be tightly regulated depending on the environmental changes to maintain homeostasis. For example, erythropoiesis repression and myelopoiesis induction are widely recognized as infectious/inflammatory conditions. Transcription factors are expected to play significant roles in achieving such alterations. However, the precise mechanism by which the differentiation trajectory shift is controlled under environmental changes has not fully been elucidated. In this study, we showed that Bach transcription factors organize the erythro-myeloid differentiation in hematopoietic stem/progenitor cells in response to the environmental changes. Under the steady state, Bach transcription factors support erythroid differentiation and repress myeloid differentiation by regulating the expression of their target genes. In contrast, the functions of Bach transcription factors were repressed, which induced myelopoiesis and repressed erythropoiesis, under emergency conditions, such as infection/inflammation. Competitive roles of Bach transcription factors and C/EBP, the key regulator of myelopoiesis, might be important for tuning erythro-myeloid differentiation trajectory and its shift in response to infection/inflammation. Disfunction of Bach transcription factors was considered to be related to anemia in chronic inflammation and myelodysplastic syndromes. In addition, considering previous findings, Bach2 might possess important roles in the conditioning of the hematopoietic system for subsequent emergency conditions.


Assuntos
Diferenciação Celular , Células-Tronco Hematopoéticas/citologia , Fatores de Transcrição/fisiologia , Fatores de Transcrição de Zíper de Leucina Básica/fisiologia , Humanos , Infecção , Inflamação , Mielopoese
9.
Rinsho Ketsueki ; 60(5): 468-474, 2019.
Artigo em Japonês | MEDLINE | ID: mdl-31168015

RESUMO

Hematopoietic stem cells (HSCs) reside at the top of the differentiation hierarchy and differentiate into multiple hematopoietic cell lineages via a finely-tuned process. HSCs have been shown to locate in a specific microenvironment (niche) that integrates HSC function, including self-renewal, and differentiation. Accumulating evidence has revealed that various types of cells in and around the bone marrow participate in HSC function regulation as niche-comprising cells. Furthermore, recent studies have identified the stromal cells closely associated with bone marrow vasculatures as HSC niche components. The remarkable advances in experimental technologies have enabled the identification of functional differences among distinct niche cell types in the bone marrow. In this article, we review recent evidence regarding the HSC niche by focusing on the cell types associated with bone marrow vasculatures and discuss future research directions.


Assuntos
Células-Tronco Hematopoéticas/citologia , Nicho de Células-Tronco , Medula Óssea , Diferenciação Celular , Humanos
10.
Biochemistry (Mosc) ; 84(3): 190-204, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31221058

RESUMO

Hematopoietic stem cells (HSCs) were the first stem cells discovered in humans. A. A. Maximov proposed an idea of blood stem cells that was confirmed later by McCulloch and Till experimentally. HSCs were the first type of stem cells to be used in clinics and ever since are being continually used. Indeed, a single HSC transplanted intravenously is capable of giving rise to all types of blood cells. In recent decades, human and animal HSC origin, development, hierarchy, and gene signature have been extensively investigated. Due to the constant need for donor blood and HSCs suitable for therapeutic transplants, the experimental possibility of obtaining HSCs in vitro by directed differentiation of pluripotent stem cells (PSCs) has been considered in recent years. However, despite all efforts, it is not yet possible to reproduce in vitro the ontogenesis of HSCs and obtain cells capable of long-term maintenance of hematopoiesis. The study of hematopoiesis in embryonic development facilitates the establishment and improvement of protocols for deriving blood cells from PCSs and allows a better understanding of the pathogenesis of various types of proliferative blood diseases, anemia, and immunodeficiency. This review focuses on the development of hematopoiesis in mammalian ontogenesis.


Assuntos
Embrião de Mamíferos/citologia , Células-Tronco Hematopoéticas/citologia , Animais , Diferenciação Celular , Humanos
11.
Immunity ; 50(6): 1439-1452.e5, 2019 06 18.
Artigo em Inglês | MEDLINE | ID: mdl-31178352

RESUMO

Hematopoietic stem cells (HSCs) are generated from specialized endothelial cells of the embryonic aorta. Inflammatory factors are implicated in regulating mouse HSC development, but which cells in the aorta-gonad-mesonephros (AGM) microenvironment produce these factors is unknown. In the adult, macrophages play both pro- and anti-inflammatory roles. We sought to examine whether macrophages or other hematopoietic cells found in the embryo prior to HSC generation were involved in the AGM HSC-generative microenvironment. CyTOF analysis of CD45+ AGM cells revealed predominance of two hematopoietic cell types, mannose-receptor positive macrophages and mannose-receptor negative myeloid cells. We show here that macrophage appearance in the AGM was dependent on the chemokine receptor Cx3cr1. These macrophages expressed a pro-inflammatory signature, localized to the aorta, and dynamically interacted with nascent and emerging intra-aortic hematopoietic cells (IAHCs). Importantly, upon macrophage depletion, no adult-repopulating HSCs were detected, thus implicating a role for pro-inflammatory AGM-associated macrophages in regulating the development of HSCs.


Assuntos
Diferenciação Celular , Desenvolvimento Embrionário , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Macrófagos/metabolismo , Animais , Biomarcadores , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Imunofluorescência , Imunofenotipagem , Inflamação/etiologia , Inflamação/metabolismo , Macrófagos/citologia , Macrófagos/imunologia , Camundongos , Camundongos Transgênicos , Células Mieloides/citologia , Células Mieloides/metabolismo
12.
Nat Commun ; 10(1): 2427, 2019 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-31160593

RESUMO

Enhancer of zeste homolog 2 (EZH2)-mediated trimethylation of histone 3 lysine 27 (H3K27Me3) is critical for immune regulation. However, evidence is lacking to address the effect of EZH2 enzyme's activity on intestinal immune responses during inflammatory bowel disease (IBD). Here we report that suppressing EZH2 activity ameliorates experimental intestinal inflammation and delayed the onset of colitis-associated cancer. In addition, we identified an increased number of functional MDSCs in the colons, which are essential for EZH2 inhibitor activity. Moreover, inhibition of EZH2 activity promotes the generation of MDSCs from hematopoietic progenitor cells in vitro, demonstrating a previously unappreciated role for EZH2 in the development of MDSCs. Together, these findings suggest the feasibility of EZH2 inhibitor clinical trials for the control of IBD. In addition, this study identifies MDSC-promoting effects of EZH2 inhibitors that may be undesirable in other therapeutic contexts and should be addressed in a clinical trial setting.


Assuntos
Colite/imunologia , Colo/imunologia , Proteína Potenciadora do Homólogo 2 de Zeste/imunologia , Doenças Inflamatórias Intestinais/imunologia , Células Supressoras Mieloides/imunologia , Animais , Diferenciação Celular/efeitos dos fármacos , Colite/induzido quimicamente , Colite/complicações , Colite/patologia , Colo/efeitos dos fármacos , Colo/patologia , Neoplasias do Colo/etiologia , Sulfato de Dextrana/toxicidade , Proteína Potenciadora do Homólogo 2 de Zeste/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Feminino , Células-Tronco Hematopoéticas/citologia , Código das Histonas , Histonas/metabolismo , Técnicas In Vitro , Indazóis/farmacologia , Indóis/farmacologia , Metilação , Camundongos , Células Supressoras Mieloides/citologia , Piridonas/farmacologia
13.
Nature ; 571(7763): 117-121, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31142833

RESUMO

Multipotent self-renewing haematopoietic stem cells (HSCs) regenerate the adult blood system after transplantation1, which is a curative therapy for numerous diseases including immunodeficiencies and leukaemias2. Although substantial effort has been applied to identifying HSC maintenance factors through the characterization of the in vivo bone-marrow HSC microenvironment or niche3-5, stable ex vivo HSC expansion has previously been unattainable6,7. Here we describe the development of a defined, albumin-free culture system that supports the long-term ex vivo expansion of functional mouse HSCs. We used a systematic optimization approach, and found that high levels of thrombopoietin synergize with low levels of stem-cell factor and fibronectin to sustain HSC self-renewal. Serum albumin has long been recognized as a major source of biological contaminants in HSC cultures8; we identify polyvinyl alcohol as a functionally superior replacement for serum albumin that is compatible with good manufacturing practice. These conditions afford between 236- and 899-fold expansions of functional HSCs over 1 month, although analysis of clonally derived cultures suggests that there is considerable heterogeneity in the self-renewal capacity of HSCs ex vivo. Using this system, HSC cultures that are derived from only 50 cells robustly engraft in recipient mice without the normal requirement for toxic pre-conditioning (for example, radiation), which may be relevant for HSC transplantation in humans. These findings therefore have important implications for both basic HSC research and clinical haematology.


Assuntos
Técnicas de Cultura de Células/métodos , Autorrenovação Celular/efeitos dos fármacos , Transplante de Células-Tronco Hematopoéticas/métodos , Células-Tronco Hematopoéticas/citologia , Animais , Proliferação de Células/efeitos dos fármacos , Células Clonais/citologia , Células Clonais/efeitos dos fármacos , Meios de Cultura/química , Meios de Cultura/farmacologia , Feminino , Fibronectinas/farmacologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Masculino , Camundongos , Álcool de Polivinil/farmacologia , Albumina Sérica , Fator de Células-Tronco/farmacologia , Trombopoetina/farmacologia , Fatores de Tempo , Condicionamento Pré-Transplante
14.
Methods Mol Biol ; 1975: 239-249, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31062313

RESUMO

Hematopoietic stem cells (HSCs) reside at the apex of the hematopoietic hierarchy, possessing the ability to self-renew and differentiate toward all mature blood lineages. Along with more specialized progenitor cells, HSCs have an essential role in maintaining a healthy blood system. Incorrect regulation of cell fate decisions in stem/progenitor cells can lead to an imbalance of mature blood cell populations-a situation seen in diseases such as leukemia. Transcription factors, acting as part of complex regulatory networks, are known to play an important role in regulating hematopoietic cell fate decisions. Yet, discovering the interactions present in these networks remains a big challenge. Here, we discuss a computational method that uses single-cell gene expression data to reconstruct Boolean gene regulatory network models and show how this technique can be applied to enhance our understanding of transcriptional regulation in hematopoiesis.


Assuntos
Diferenciação Celular , Linhagem da Célula , Biologia Computacional/métodos , Redes Reguladoras de Genes , Hematopoese , Células-Tronco Hematopoéticas/citologia , Humanos , Transcriptoma
15.
Cryo Letters ; 40(3): 181-186, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31095667

RESUMO

BACKGROUND: Cryopreservation of hematopoietic stem cells (HSCs) has widely been used in stem cell transplantation and cellular therapy treating various human diseases. However, the current conventional cooling approach for the cryopreservation of HSCs has the following potential problems: (1) requirement of a very expensive computer-programmed liquid nitrogen freezer (LNF) for the cooling rate control, (2) a large consumption of liquid nitrogen, (3) periodic breakdown of the LNF due to the mechanical failure of the liquid nitrogen valves (i.e., magnetic-solenoid valves) inside the LNF, and (4) constant monitoring of the LNF operation during the HSCs cooling process. OBJECTIVE: To test and evaluate a simple and reliable approach for the cryopreservation of HSCs using the passive cooling technique. MATERIALS AND METHODS: A passive cooling-rate-controlled device (PCD) was developed and used to cryopreserve HSCs. The PCD is inexpensive, simple, and user-friendly, which needs only the minimum maintenance and no consumption of liquid nitrogen. The PCD was compared to the LNF for the cryopreservation of HSCs in the present study through experiments. The cell viability and functionality were evaluated after cryopreservation. RESULTS: In comparison with the LNF method, the PCD approach enabled high cell viability/survival, recovery rate, and functionality after cryopreservation processes. CONCLUSION: The PCD offers a cost-effective, simple, and reliable approach for the optimal cryopreservation of HSCs.


Assuntos
Criopreservação/métodos , Células-Tronco Hematopoéticas/citologia , Sobrevivência Celular , Congelamento , Humanos
16.
Nat Cell Biol ; 21(6): 700-709, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31061465

RESUMO

Haematopoietic stem cells (HSCs) maintain balanced self-renewal and differentiation, but how these functions are precisely regulated is not fully understood. N6-methyladenosine (m6A) messenger RNA methylation has emerged as an important mode of epitranscriptional gene expression regulation affecting many biological processes. We show that deletion of the m6A methyltransferase Mettl3 from the adult haematopoietic system led to an accumulation of HSCs in the bone marrow and a marked reduction of reconstitution potential due to a blockage of HSC differentiation. Interestingly, deleting Mettl3 from myeloid cells using Lysm-cre did not impact myeloid cell number or function. RNA sequencing revealed 2,073 genes with significant m6A modifications in HSCs. Myc was identified as a direct target of m6A in HSCs. Mettl3-deficient HSCs failed to upregulate MYC expression following stimulation to differentiate and enforced expression of Myc rescued differentiation defects of Mettl3-deficient HSCs. Our results reveal a key role of m6A in governing HSC differentiation.


Assuntos
Adenosina/análogos & derivados , Diferenciação Celular/genética , Células-Tronco Hematopoéticas/citologia , Metiltransferases/genética , Proteínas Proto-Oncogênicas c-myc/genética , Adenosina/genética , Animais , Células da Medula Óssea/citologia , Células da Medula Óssea/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/genética , Células-Tronco Hematopoéticas/metabolismo , Metilação , Camundongos , RNA Mensageiro/genética , Análise de Sequência de RNA
17.
Nat Cell Biol ; 21(6): 721-730, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31110287

RESUMO

Wnt signalling drives many processes in development, homeostasis and disease; however, the role and mechanism of individual ligand-receptor (Wnt-Frizzled (Fzd)) interactions in specific biological processes remain poorly understood. Wnt9a is specifically required for the amplification of blood progenitor cells during development. Using genetic studies in zebrafish and human embryonic stem cells, paired with in vitro cell biology and biochemistry, we determined that Wnt9a signals specifically through Fzd9b to elicit ß-catenin-dependent Wnt signalling that regulates haematopoietic stem and progenitor cell emergence. We demonstrate that the epidermal growth factor receptor (EGFR) is required as a cofactor for Wnt9a-Fzd9b signalling. EGFR-mediated phosphorylation of one tyrosine residue on the Fzd9b intracellular tail in response to Wnt9a promotes internalization of the Wnt9a-Fzd9b-LRP signalosome and subsequent signal transduction. These findings provide mechanistic insights for specific Wnt-Fzd signals, which will be crucial for specific therapeutic targeting and regenerative medicine.


Assuntos
Células-Tronco Hematopoéticas/citologia , Receptores de Neurotransmissores/genética , Proteínas Wnt/genética , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/genética , Animais , Receptores ErbB/genética , Humanos , Fosforilação , Via de Sinalização Wnt , Peixe-Zebra/crescimento & desenvolvimento , beta Catenina/genética
18.
Int J Mol Sci ; 20(7)2019 Apr 08.
Artigo em Inglês | MEDLINE | ID: mdl-30965622

RESUMO

Although human pluripotent stem cells (hPSCs) can theoretically differentiate into any cell type, their ability to produce hematopoietic cells is highly variable from one cell line to another. The underlying mechanisms of this heterogeneity are not clearly understood. Here, using a whole miRNome analysis approach in hPSCs, we discovered that their hematopoietic competency was associated with the expression of several miRNAs and conversely correlated to that of miR-206 specifically. Lentiviral-based miR-206 ectopic expression in H1 hematopoietic competent embryonic stem (ES) cells markedly impaired their differentiation toward the blood lineage. Integrative bioinformatics identified a potential miR-206 target gene network which included hematopoietic master regulators RUNX1 and TAL1. This work sheds light on the critical role of miR-206 in the generation of blood cells off hPSCs. Our results pave the way for future genetic manipulation of hPSCs aimed at increasing their blood regenerative potential and designing better protocols for the generation of bona fide hPSC-derived hematopoietic stem cells.


Assuntos
MicroRNAs/metabolismo , Células-Tronco Pluripotentes/citologia , Diferenciação Celular/fisiologia , Linhagem Celular , Linhagem da Célula , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Humanos , Células-Tronco Pluripotentes/metabolismo
19.
Int J Mol Sci ; 20(8)2019 Apr 23.
Artigo em Inglês | MEDLINE | ID: mdl-31018542

RESUMO

Cord blood (CB) is an attractive source of hematopoietic stem cells (HSCs) for hematopoietic cell transplantation. However, its application remains limited due to the low number of HSCs/progenitors in a single CB unit and its notoriously difficulty in expanding ex vivo. Here, we demonstrated that the human fetal liver sinusoidal endothelial cells engineered to constitutively express the adenoviral E4orf1 gene (hFLSECs-E4orf1) is capable of efficient expansion ex vivo for human CB hematopoietic stem and progenitor cells (HSPCs). Coculture of CD34+ hCB cells with hFLSECs-E4orf1 resulted in generation of substantially more total nucleated cells, CD34+CD38- and CD34+ CD38-CD90+ HSPCs in comparison with that of cytokines alone after 14 days. The multilineage differentiation potential of the expanded hematopoietic cells in coculture condition, as assessed by in vitro colony formation, was also significantly heightened. The CD34+ hCB cells amplified on hFLSECs-E4orf1 were capable of engraftment in vivo. Furthermore, hFLSECs-E4orf1 highly expressed hematopoiesis related growth factor and Notch receptors. Accordingly, the CD34+ hCB cells amplified on hFLSECs-E4orf1 exhibited Notch signaling activation. Taken together, our findings indicated that FLSECs may potentially be the crucial component of the microenvironment to support recapitulation of embryonic HSC amplification in vitro and allow identification of new growth factors responsible for collective regulation of hematopoiesis.


Assuntos
Técnicas de Cocultura/métodos , Células Endoteliais/citologia , Células-Tronco Hematopoéticas/citologia , Fígado/citologia , Animais , Antígenos CD34/análise , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Hematopoese , Transplante de Células-Tronco Hematopoéticas , Humanos , Fígado/embriologia , Camundongos
20.
Int J Mol Sci ; 20(7)2019 Apr 11.
Artigo em Inglês | MEDLINE | ID: mdl-30978983

RESUMO

The bone marrow (BM) microenvironment plays a crucial role in the maintenance and regeneration of hematopoietic stem (HSC) and progenitor cells (HSPC). In particular, the vascular niche is responsible for regulating HSC maintenance, differentiation, and migration of cells in and out of the BM. Damage to this niche upon exposure to ionizing radiation, whether accidental or as a result of therapy, can contribute to delays in HSC recovery and/or function. The ability of BM derived-endothelial cells (BMEC) to alter and/or protect HSPC after exposure to ionizing radiation was investigated. Our data show that exposure of BMEC to ionizing radiation resulted in alterations in Akt signaling, increased expression of PARP-1, IL6, and MCP-1, and decreased expression of MMP1 and MMP9. In addition, global analysis of gene expression of HSC and BMEC in response to mixed neutron/gamma field (MF) radiation identified 60 genes whose expression was altered after radiation in both cell types, suggesting that a subset of genes is commonly affected by this type of radiation. Focused gene analysis by RT-PCR revealed two categories of BMEC alterations: (a) a subset of genes whose expression was altered in response to radiation, with no additional effect observed during coculture with HSPC, and (b) a subset of genes upregulated in response to radiation, and altered when cocultured with HSPC. Coculture of BMEC with CD34+ HSPC induced HSPC proliferation, and improved BM function after MF radiation. Nonirradiated HSPC exhibited reduced CD34 expression over time, but when irradiated, they maintained higher CD34 expression. Nonirradiated HSPC cocultured with nonirradiated BMEC expressed lower levels of CD34 expression compared to nonirradiated alone. These data characterize the role of each cell type in response to MF radiation and demonstrate the interdependence of each cell's response to ionizing radiation. The identified genes modulated by radiation and coculture provide guidance for future experiments to test hypotheses concerning specific factors mediating the beneficial effects of BMEC on HSPC. This information will prove useful in the search for medical countermeasures to radiation-induced hematopoietic injury.


Assuntos
Células da Medula Óssea/efeitos da radiação , Técnicas de Cocultura , Células Endoteliais/efeitos da radiação , Células-Tronco Hematopoéticas/efeitos da radiação , Antígenos CD34/análise , Células da Medula Óssea/citologia , Células da Medula Óssea/metabolismo , Diferenciação Celular/efeitos da radiação , Linhagem Celular , Proliferação de Células/efeitos da radiação , Técnicas de Cocultura/métodos , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Raios gama , Regulação da Expressão Gênica/efeitos da radiação , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Humanos , Nêutrons , Fenótipo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Lesões por Radiação/prevenção & controle , Transdução de Sinais/efeitos da radiação
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