Zebrafish kidney marrow contains ABCG2-dependent side population cells exhibiting hematopoietic stem cell properties

Zebrafish (Danio rerio) has emerged as a powerful genetic model for the study of vertebrate hematopoiesis. However, methods for detection and isolation of hematopoietic stem cells (HSCs) have not yet been reported. In mammals, the combination of Hoechst 33342 staining with flow cytometry can be used for separation of a bone marrow side population (SP), which is highly enriched for HSCs. We applied a similar procedure to hematopoietic kidney marrow cells from adult zebrafish, and identified a segregated cohort of SP cells, which demonstrate a set of features typical of stem cells. SP cells show extremely low scatter characteristics, and are small in size with a minimum of cytoplasm. Treatment of zebrafish kidney marrow cells with reserpine or fumitremorgin C, which inhibit the ABCG2 transporter responsible for Hoechst 33342 efflux, caused a clear reduction in the number of SP cells. Consistent with the quiescent state of HSCs, the SP cells are strongly resistant to the myelosuppressive agent 5-fluorouracil. In addition, SP cells specifically demonstrate higher expression of genes known to be markers of HSCs of mammals. Hence, our results show that the SP phenotype is conserved between mammals and teleosts, and the properties of the zebrafish SP cells indicate a significant enrichment for HSCs. These rapid flow cytometric methods for purification of HSCs from zebrafish may greatly facilitate genetic analysis of stem cells using the advantages of this vertebrate model.     

Quantitative and qualitative in vitro analysis of the stem cell potential of hematopoietic cells purified from murine skeletal muscle

The murine skeletal muscle contains hematopoietic stem cells, but this potential has so far not been studied quantitatively or qualitatively in vitro. To quantify the hematopoietic stem cell potential, we have used highly purified SP/CD45^＋ cells in long-term culture initiating cell （LTC-IC） assays. The SP/CD45^＋ cell population purified from murine muscle was found to have significant stem cell activity with an LTC-IC frequency of 1/640. Single-cell-sorted SP/CD45^＋ cells from muscle exhibited robust proliferative activity in vitro at day 16 （380-fold amplification）, especially after culture with OP-9 layers that also support embryonic stem cells. Amplified cell populations originating from single cells exhibited multilineage differentiation ability with evidence of myeloid, lymphoid and NK cell markers. Thus, our results demonstrate that hematopoietic stem cells that can be quantified by LTC-IC assays exist in the murine skeletal muscle and show also for the first time, at the single-cell level, that these cells exhibit multilineage differentiation ability and major proliferative potential.     

Young donor hematopoietic stem cells revitalize aged or damaged bone marrow niche by transdifferentiating into functional niche cells

The bone marrow niche maintains hematopoietic stem cell (HSC) homeostasis and declines in function in the physiologically aging population and in patients with hematological malignancies. A fundamental question is now whether and how HSCs are able to renew or repair their niche. Here, we show that disabling HSCs based on disrupting autophagy accelerated niche aging in mice, whereas transplantation of young, but not aged or impaired, donor HSCs normalized niche cell populations and restored niche factors in host mice carrying an artificially harassed niche and in physiologically aged host mice, as well as in leukemia patients. Mechanistically, HSCs, identified using a donor lineage fluorescence-tracing system, transdifferentiate in an autophagy-dependent manner into functional niche cells in the host that include mesenchymal stromal cells and endothelial cells, previously regarded as “nonhematopoietic” sources. Our findings thus identify young donor HSCs as a primary parental source of the niche, thereby suggesting a clinical solution to revitalizing aged or damaged bone marrow hematopoietic niche.     

Zebrafish adult pigment stem cells are multipotent and form pigment cells by a progressive fate restriction process

Skin pigment pattern formation is a paradigmatic example of pattern formation. In zebrafish, the adult body stripes are generated by coordinated rearrangement of three distinct pigment cell‐types, black melanocytes, shiny iridophores and yellow xanthophores. A stem cell origin of melanocytes and iridophores has been proposed although the potency of those stem cells has remained unclear. Xanthophores, however, seemed to originate predominantly from proliferation of embryonic xanthophores. Now, data from Singh et al. shows that all three cell‐types derive from shared stem cells, and that these cells generate peripheral neural cell‐types too. Furthermore, clonal compositions are best explained by a progressive fate restriction model generating the individual cell‐types. The numbers of adult pigment stem cells associated with the dorsal root ganglia remain low, but progenitor numbers increase significantly during larval development up to metamorphosis, likely via production of partially restricted progenitors on the spinal nerves.     

间充质干细胞促进脐带血干细胞体外扩增的研究进展

非亲缘脐带血移植是治疗造血系统疾病的重要移植方式之一,但脐带血移植面临的最大挑战是造血干细胞(HSCs)数量不足,特别是成人患者受到脐带血干细胞数量的限制,导致造血及免疫恢复延迟,非复发死亡率升高。体外扩增脐带血HSCs(UCB-HSCs)是解决该问题的途径之一。研究发现可以通过模拟骨髓造血龛(niche)这一生态位使HSCs在体外进行自我更新增殖,而间充质干细胞(MSCs)正是造血龛的重要的组成细胞之一。本文将探讨MSCs在UCB-HSCs体外扩增中的应用。重点以MSCs促造血的特点、机制,促进脐带血干细胞增殖的各种策略以及其临床应用和前景做一综述。     

Homologous Recombination in Zebrafish ES Cells

Targeted insertion of a plasmid by homologous recombination was demonstrated in zebrafish ES cell cultures. Two selection strategies were used to isolate ES cell colonies that contained targeted plasmid insertions in either the no tail or myostatin I gene. One selection strategy involved the manual isolation of targeted cell colonies that were identified by the loss of fluorescent protein gene expression. A second strategy used the diphtheria toxin A-chain gene in a positive-negative selection approach. Homologous recombination was confirmed by PCR, sequence and Southern blot analysis and colonies isolated using both selection methods were expanded and maintained for multiple passages. The results demonstrate that zebrafish ES cells have potential for use in a cell-mediated gene targeting approach.     

Cell–cell interaction networks regulate blood stem and progenitor cell fate

Communication networks between cells and tissues are necessary for homeostasis in multicellular organisms. Intercellular (between cell) communication networks are particularly relevant in stem cell biology, as stem cell fate decisions (self‐renewal, proliferation, lineage specification) are tightly regulated based on physiological demand. We have developed a novel mathematical model of blood stem cell development incorporating cell‐level kinetic parameters as functions of secreted molecule‐mediated intercellular networks. By relation to quantitative cellular assays, our model is capable of predictively simulating many disparate features of both normal and malignant hematopoiesis, relating internal parameters and microenvironmental variables to measurable cell fate outcomes. Through integrated in silico and experimental analyses, we show that blood stem and progenitor cell fate is regulated by cell–cell feedback, and can be controlled non‐cell autonomously by dynamically perturbing intercellular signalling. We extend this concept by demonstrating that variability in the secretion rates of the intercellular regulators is sufficient to explain heterogeneity in culture outputs, and that loss of responsiveness to cell–cell feedback signalling is both necessary and sufficient to induce leukemic transformation in silico.     

Common origins of blood and blood vessels in adults?

After embryonic development, the vast majority of cells are differentiated and all organs are in place. Growth of the organism then ensues and continues until adulthood, whereupon cell division largely ceases. In some tissues, notably the bone marrow, skin, and gut, cell proliferation continues throughout life to replace cells lost by attrition. This regeneration is fueled by rare, long-lived, and largely quiescent stem cells that give rise to committed progenitors, which in turn generate large numbers of fully differentiated cells. Mounting evidence suggests that such cells can significantly contribute to tissue repair and regeneration in adults and may therefore prove beneficial for autologous cell and gene therapies. This review focuses on the potential of adult stem cells to give rise to hematopoietic and vascular cells. We discuss evidence that a highly purified population of adult stem cells, termed SP cells, serves as a hematopoietic progenitor and can contribute to vascular regeneration after injury. We also discuss the potential relationship of these cells to the embryonic hemangioblast.     

胚胎干细胞向血液干细胞定向分化的研究进展

骨髓移植是目前治疗恶性白血病以及遗传性血液病最有效的方法之一。但是HLA相匹配的骨髓捐献者严重短缺,骨髓造血干细胞（hematopoietic stem cells,HSCs）体外培养困难,在体外修复患者骨髓造血干细胞技术不成熟,这些都大大限制了骨髓移植在临床上的应用。多能性胚胎干细胞（embryonic stem cells,ESCs）具有自我更新能力,在合适的培养条件下分化形成各种血系细胞,是造血干细胞的另一来源。在过去的二十多年里,血发生的研究是干细胞生物学中最为活跃的领域之一。小鼠及人的胚胎干细胞方面的研究最近取得了重大进展。这篇综述总结了近年来从胚胎干细胞获得造血干细胞的成就,以及在安全和技术上的障碍。胚胎干细胞诱导生成可移植性血干细胞的研究能够使我们更好地了解正常和异常造血发生的机制,同时也为造血干细胞的临床应用提供理论和实验依据。     

Mitophagy in hematopoietic stem cells

Hematopoietic stem cells (HSCs) are inherently quiescent and self-renewing, yet can differentiate and commit to multiple blood cell types. Intracellular mitochondrial content is dynamic, and there is an increase in mitochondrial content during differentiation and lineage commitment in HSCs. HSCs reside in a hypoxic niche within the bone marrow and rely heavily on glycolysis, while differentiated and committed progenitors rely on oxidative phosphorylation. Increased oxidative phosphorylation during differentiation and commitment is not only due to increased mitochondrial content but also due to changes in mitochondrial cytosolic distribution and efficiency. These changes in the intracellular mitochondrial landscape contribute signals toward regulating differentiation and commitment. Thus, a functional relationship exists between the mitochondria in HSCs and the state of the HSCs (i.e., stemness vs. differentiated). This review focuses on how autophagy-mediated mitochondrial clearance (i.e., mitophagy) may affect HSC mitochondrial content, thereby influencing the fate of HSCs and maintenance of hematopoietic homeostasis.     

胚胎干细胞向造血细胞分化研究

胚胎干（embryonic stem,ES）细胞是来源于囊胚的内细胞团（inner cell mass,ICM）,具有发育的全能性或多能性,能嵌合到早期胚胎,在体内可以参与各种组织发育甚至包括生殖细胞;在体外分化培养条件下,可以顺序分化出各种组织细胞,与体内完整胚胎发育过程相符合,而且可以通过调节ES细胞某些基因的表达而调节其分化。因此,ES细胞是研究哺乳动物早期胚胎发育、细胞分化及其关键基因鉴定的理想模型。另外,胚胎生殖脊（embryonic germ,EG）细胞系也具有同样的生物学特性,它是由早期胚胎的原始生殖脊（primordial germ,PG）细胞建株而来。最近研究显示：ES细胞在体外不但可以分化为所有造血细胞系,而且还可以分化为具有长期增殖能力的造血干细胞。作者就胚胎干细胞向造血细胞和造血干细胞分化及其诱导因子和调控基因的表达作一综述。     

Autophagy in stem cells

Autophagy is a highly conserved cellular process by which cytoplasmic components are sequestered in autophagosomes and delivered to lysosomes for degradation. As a major intracellular degradation and recycling pathway, autophagy is crucial for maintaining cellular homeostasis as well as remodeling during normal development, and dysfunctions in autophagy have been associated with a variety of pathologies including cancer, inflammatory bowel disease and neurodegenerative disease. Stem cells are unique in their ability to self-renew and differentiate into various cells in the body, which are important in development, tissue renewal and a range of disease processes. Therefore, it is predicted that autophagy would be crucial for the quality control mechanisms and maintenance of cellular homeostasis in various stem cells given their relatively long life in the organisms. In contrast to the extensive body of knowledge available for somatic cells, the role of autophagy in the maintenance and function of stem cells is only beginning to be revealed as a result of recent studies. Here we provide a comprehensive review of the current understanding of the mechanisms and regulation of autophagy in embryonic stem cells, several tissue stem cells (particularly hematopoietic stem cells), as well as a number of cancer stem cells. We discuss how recent studies of different knockout mice models have defined the roles of various autophagy genes and related pathways in the regulation of the maintenance, expansion and differentiation of various stem cells. We also highlight the many unanswered questions that will help to drive further research at the intersection of autophagy and stem cell biology in the near future.     

Methods to produce induced pluripotent stem cell-derived mesenchymal stem cells: Mesenchymal stem cells from induced pluripotent stem cells

Mesenchymal stem cells (MSCs) have received significant attention in recent years due to their large potential for cell therapy. Indeed, they secrete a wide variety of immunomodulatory factors of interest for the treatment of immune-related disorders and inflammatory diseases. MSCs can be extracted from multiple tissues of the human body. However, several factors may restrict their use for clinical applications: the requirement of invasive procedures for their isolation, their limited numbers, and their heterogeneity according to the tissue of origin or donor. In addition, MSCs often present early signs of replicative senescence limiting their expansion in vitro, and their therapeutic capacity in vivo. Due to the clinical potential of MSCs, a considerable number of methods to differentiate induced pluripotent stem cells (iPSCs) into MSCs have emerged. iPSCs represent a new reliable, unlimited source to generate MSCs (MSCs derived from iPSC, iMSCs) from homogeneous and well-characterized cell lines, which would relieve many of the above mentioned technical and biological limitations. Additionally, the use of iPSCs prevents some of the ethical concerns surrounding the use of human embryonic stem cells. In this review, we analyze the main current protocols used to differentiate human iPSCs into MSCs, which we classify into five different categories: MSC Switch, Embryoid Body Formation, Specific Differentiation, Pathway Inhibitor, and Platelet Lysate. We also evaluate common and method-specific culture components and provide a list of positive and negative markers for MSC characterization. Further guidance on material requirements to produce iMSCs with these methods and on the phenotypic features of the iMSCs obtained is added. The information may help researchers identify protocol options to design and/or refine standardized procedures for large-scale production of iMSCs fitting clinical demands.     

Temporal molecular program of human hematopoietic stem and progenitor cells after birth

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