Niches of Hematopoietic Stem Cells in Bone Marrow

Molecular Biology - Hematopoietic stem cells (HSCs) exist in a close contact with their specific microenvironment, called a niche, which supports the HSC function and significantly influences the...     

DKK1 and Kremen Expression Predicts the Osteoblastic Response to Bone Metastasis

Bone metastasis is a complication of advanced breast and prostate cancer. Tumor-secreted Dickkopf homolog 1 (DKK1), an inhibitor of canonical Wnt signaling and osteoblast differentiation, was proposed to regulate the osteoblastic response to metastatic cancer in bone. The objectives of this study were to compare DKK1 expression with the in vivo osteoblastic response in a panel of breast and prostate cancer cell lines, and to discover mechanisms that regulate cancer DKK1 expression. DKK1 expression was highest in MDA-MB-231 and PC3 cells that produce osteolytic lesions, and hence a suppressed osteoblastic response, in animal models of bone metastasis. LnCaP, C4-2B, LuCaP23.1, T47D, ZR-75-1, MCF-7, ARCaP and ARCaP_M cancer cells that generate osteoblastic, mixed or no bone lesions had the lowest DKK1 expression. The cell lines with negligible expression, LnCaP, C4-2B and T47D, exhibited methylation of the DKK1 promoter. Canonical Wnt signaling activity was then determined and found in all cell lines tested, even in the MDA-MB-231 and PC3 cell lines despite sizeable amounts of DKK1 protein expression expected to block canonical Wnt signaling. A mechanism of DKK1 resistance in the osteolytic cell lines was investigated and determined to be at least partially due to down-regulation of the DKK1 receptors Kremen1 and Kremen2 in the MDA-MB-231 and PC3 cell lines. Combined DKK1 and Kremen expression in cancer cells may serve as predictive markers of the osteoblastic response of breast and prostate cancer bone metastasis.     

Endocannabinoids Are Expressed in Bone Marrow Stromal Niches and Play a Role in Interactions of Hematopoietic Stem and Progenitor Cells with the Bone Marrow Microenvironment

Endocannabinoids are lipid signaling molecules that act via G-coupled receptors, CB₁ and CB₂. The endocannabinoid system is capable of activation of distinct signaling pathways on demand in response to pathogenic events or stimuli, hereby enhancing cell survival and promoting tissue repair. However, the role of endocannabinoids in hematopoietic stem and progenitor cells (HSPCs) and their interaction with hematopoietic stem cells (HSC) niches is not known. HSPCs are maintained in the quiescent state in bone marrow (BM) niches by intrinsic and extrinsic signaling. We report that HSPCs express the CB₁ receptors and that BM stromal cells secrete endocannabinoids, anandamide (AEA) (35 pg/10⁷ cells), and 2-AG (75.2 ng/10⁷ cells). In response to the endotoxin lipopolysaccharide (LPS), elevated levels of AEA (75.6 pg/10⁷ cells) and 2-AG (98.8 ng/10⁷ cells) were secreted from BM stromal cells, resulting in migration and trafficking of HSPCs from the BM niches to the peripheral blood. Furthermore, administration of exogenous cannabinoid CB₁ agonists in vivo induced chemotaxis, migration, and mobilization of human and murine HSPCs. Cannabinoid receptor knock-out mice Cnr1^−/− showed a decrease in side population (SP) cells, whereas fatty acid amide hydrolase (FAAH)^−/− mice, which have elevated levels of AEA, yielded increased colony formation as compared with WT mice. In addition, G-CSF-induced mobilization in vivo was modulated by endocannabinoids and was inhibited by specific cannabinoid antagonists as well as impaired in cannabinoid receptor knock-out mice Cnr1^−/−, as compared with WT mice. Thus, we propose a novel function of the endocannabinoid system, as a regulator of HSPC interactions with their BM niches, where endocannabinoids are expressed in HSC niches and under stress conditions, endocannabinoid expression levels are enhanced to induce HSPC migration for proper hematopoiesis.     

Overexpression of FGF9 in Prostate Epithelial Cells Augments Reactive Stroma Formation and Promotes Prostate Cancer Progression

Bone metastasis is the major cause of morbidity and mortality of prostate cancer (PCa). Fibroblast growth factor 9 (FGF9) has been reported to promote PCa bone metastasis. However, the mechanism by which overexpression of FGF9 promotes PCa progression and metastasis is still unknown. Herein, we report that transgenic mice forced to express FGF9 in prostate epithelial cells (F9TG) developed high grade prostatic intraepithelial neoplasia (PIN) in an expression level- and time-dependent manner. Moreover, FGF9/TRAMP bigenic mice (F9TRAMP) grew advanced PCa earlier and had higher frequencies of metastasis than TRAMP littermates. We observed tumor microenvironmental changes including hypercellularity and hyperproliferation in the stromal compartment of F9TG and F9TRAMP mice. Expression of TGFβ1, a key signaling molecule overexpressed in reactive stroma, was increased in F9TG and F9TRAMP prostates. Both in vivo and in vitro data indicated that FGF9 promoted TGFβ1 expression via increasing cJun-mediated signaling. Moreover, in silico analyses showed that the expression level of FGF9 was positively associated with expression of TGFβ1 and its downstream signaling molecules in human prostate cancers. Collectively, our data demonstrated that overexpressing FGF9 in PCa cells augmented the formation of reactive stroma and promoted PCa initiation and progression.     

Hematopoietic Stem Cell Heterogeneity Is Linked to the Initiation and Therapeutic Response of Myeloproliferative Neoplasms

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PSCA与前列腺癌研究进展

前列腺干细胞抗原(prostate stem cell antigen,PSCA)是一种前列腺癌相关肿瘤抗原,也是一种GPI(gly-cosyl phosphatidylinositol)锚定蛋白,通过其C端的GPI锚定结构锚定到细胞膜表面.PSCA在正常前列腺组织中的表达较低,提高的PSCA表达伴随着增加的肿瘤分期、分级以及雄激素非依赖性和转移癌的形成,且不随癌症进展而降低,是前列腺癌诊断和治疗的理想靶抗原.动物实验显示,PSCA抗体和疫苗可能在前列腺癌免疫靶向治疗中具有重要价值.     

Thrombopoietin-Increased DNA-PK-Dependent DNA Repair Limits Hematopoietic Stem and Progenitor Cell Mutagenesis in Response to DNA Damage

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Highlights? Mpl loss increases γ-irradiation-induced genomic instability in HSPCs ? TPO promotes DNA repair in vitro and in vivo in HSPCs ? TPO increases DNA-PK activity and NHEJ-mediated repair efficiency in HSPCs ? A single TPO injection before mouse TBI limits long-term HSC injury and mutagenesis     

Gadd45a基因对小鼠造血干细胞功能的影响

目的 Gadd45a基因对小鼠造血干细胞功能的影响。方法流式细胞仪分选小鼠骨髓造血干细胞、体外单克隆培养,竞争性骨髓移植,放射线照射观察生存曲线。结果 Gadd45a基因缺失的小鼠造血干细胞克隆形成能力增强,短期造血重建能力无差异,8.5Gy放射线照射后生存情况无差异。结论 Gadd45a基因对小鼠造血干细胞功能起重要作用。     

Regulation of Cell Cycle in Hematopoietic Stem Cells by the Niche

The quiescent state is thought to be an indispensable property for themaintenance of hematopoietic stem cells (HSCs). Interaction of HSCs with theirparticular microenvironments, known as the stem cell niches, is critical for cell cycleregulation of HSCs. Monitoring of the quiescence of HSCs using by a new stem cellmarker, Side Population (SP), revealed that the cell cycle status of HSCs is dynamicallycontrolled by the microenvironments. We have recently revealed a molecularmechanism in which cell cycle of HSCs is regulated by the niche. HSCs expressing thereceptor tyrosine kinase Tie2 are adhere to osteoblasts (OBs) in the BM niche. Theinteraction of Tie2 and its ligand Angiopoietin-1 (Ang-1) leads to tight adhesion ofHSCs to stromal cells, resulting in maintainance of long-term repopulating activity ofHSCs. Thus, Tie2/Ang-1 signaling pathway plays a critical role in the maintenance ofHSCs in a quiescent state in the BM niche. The understanding of cell cycle control instem cells leads to development of new strategy for progress in regenerative medicine.     

Regulation of Epithelial Stem Cell Replacement and Follicle Formation in the Drosophila Ovary

Though much has been learned about the process of ovarian follicle maturation through studies of oogenesis in both vertebrate and invertebrate systems, less is known about how follicles form initially. In Drosophila, two somatic follicle stem cells (FSCs) in each ovariole give rise to all polar cells, stalk cells, and main body cells needed to form each follicle. We show that one daughter from each FSC founds most follicles but that cell type specification is independent of cell lineage, in contrast to previous claims of an early polar/stalk lineage restriction. Instead, key intercellular signals begin early and guide cell behavior. An initial Notch signal from germ cells is required for FSC daughters to migrate across the ovariole and on occasion to replace the opposite stem cell. Both anterior and posterior polar cells arise in region 2b at a time when ∼16 cells surround the cyst. Later, during budding, stalk cells and additional polar cells are specified in a process that frequently transfers posterior follicle cells onto the anterior surface of the next older follicle. These studies provide new insight into the mechanisms that underlie stem cell replacement and follicle formation during Drosophila oogenesis.THE Drosophila ovary is a highly favorable system for studying epithelial cell differentiation downstream from a stem cell (reviewed in Blanpain et al. 2007; Kirilly and Xie 2007). New follicles consisting of 16 interconnected germ cells surrounded by an epithelial (follicle cell) monolayer are continuously produced during adult life and develop sequentially within ovarioles (reviewed in Spradling 1993). Follicle formation begins in the germarium (Figure 1A), a structure at the tip of each ovariole that houses 2–3 germline stem cells (GSCs) and 2 follicle stem cells (FSCs) within stable niches (reviewed in Morrison and Spradling 2008). Successive GSC daughters known as cystoblasts are enclosed by a thin covering of squamous escort cells and divide asymmetrically four times in sucession to produce 16-cell germline cysts, comprising 15 presumptive nurse cells and a presumptive oocyte (reviewed in de Cuevas et al. 1997). At the junction between region 2a and region 2b, cysts are forced into single file as they encounter the FSCs, lose their escort cell covering, and begin to acquire a follicular layer. Follicle cells derived from both FSCs soon mold them into a “lens shape” characteristic of region 2b. Under the influence of continued somatic cell growth, cysts and their surrounding cells round up, enter region 3 (also known as stage 1), and bud from the germarium as new follicles that remain connected to their neighbors by short cellular stalks (Figure 1B).Open in a separate windowFigure 1.—Prefollicle cells associate with cysts in an ordered fashion downstream from the FSCs. (A) A diagram of the Drosophila germarium showing the four subregions: 1, 2a, 2b, and 3. Two GSCs (orange) reside in region 1 and produce cysts (yellow ovals). Two FSCs reside at the border of regions 2a and 2b and produce follicle cells that encapsulate region 2b and region 3 cysts. (B) A diagram of two follicles that have budded from the germarium showing their pairs of anterior and posterior polar cells as well as the interconnecting 4–6 stalk cells. (C) Germaria stained with anti-traffic jam (green) to mark somatic cells, anti-vasa (red) to mark germ cells, and DAPI (blue). The numbers of somatic cells associated with each cyst (indicated) were reconstructed from three-dimensional image stacks. (D–F) Small transient clones stained with anti-LacZ (green, the clonal marker), anti-FasIII (red), and DAPI (blue). Regions 2b and 3 cysts are outlined in white. Pink dots indicate labeled FSC daughters; however, not all labeled cells are marked because some are not visible in the presented plane of focus. (D) A 4-cell clone associated with the first cyst in region 2b. (E) An 8-cell clone associated with the second region 2b cyst. (F) A 15-cell clone associated with the region 3 cyst. (G) Model of follicle layer acquisition. One FSC daughter, the cmc (light green, left) contacts the anterior face of the incoming cyst (2a/b, orange) and founds mostly anterior follicle cells (light green). Another FSC daughter, the pmc (dark green, left) contacts the posterior cyst face and founds mostly posterior follicle cells (dark green). Bar, 10 μm; anterior is to the left.A complex sequence of signaling and adhesive interactions between follicular and germline cells is required for follicle budding, oocyte development, and patterning (reviewed in Huynh and St. Johnston 2004). However, the mechanisms orchestrating the initial association between follicle cells and cysts within the germarium are less well understood. While lineage analysis indicates the presence of two FSCs (Margolis and Spradling 1995; Nystul and Spradling 2007), low fasciclin III (FasIII) expression has been claimed to specifically mark FSCs, leading to the conclusion that more FSCs are present under some conditions (Zhang and Kalderon 2001; Vied and Kalderon 2009).The differentiation of polar cells at both their anterior and posterior ends is required for normal follicle production (Ruohola et al. 1991; Larkin et al. 1996; Grammont and Irvine 2001), and depends on Notch signals received from the germline (Lopez-Schier and St. Johnston 2001). Subsequently, anterior polar cells send JAK-STAT and Notch signals that specify stalk cells (McGregor et al. 2002; Torres et al. 2003; Assa-Kunik et al. 2007). While the source of these signals and their effects are clear, the timing of polar cell specification and its dependence on cell lineage are not. Some anterior and posterior polar cells (but not stalk cells) were inferred by lineage analysis to arise and cease division within region 2b (Margolis and Spradling 1995). In contrast, on the basis of marker gene expression it was concluded that anterior polar cells are specified later, in stage 1, and posterior polar cells in stage 2 (Torres et al. 2003). Up to four polar cells may eventually form, but apoptosis reduces their number to a single pair at each end by stage 5 (Besse and Pret 2003). Moreover, polar and stalk are believed to arise exclusively from “polar/stalk” precursors that separate from the rest of the FSC lineage (Larkin et al. 1996; Tworoger et al. 1999; Besse and Pret 2003) and these cells were proposed to invade between the last region 2b cyst to affect follicle budding (Torres et al. 2003; Assa-Kunik et al. 2007).Here we have analyzed the detailed behavior of FSCs and their daughters in the germarium. No evidence of polar/stalk precursors was observed, and we show that the first anterior and posterior polar cells are specified in region 2b, prior to the previously accepted time of follicle cell specialization. Additional polar cells are also formed later during stages 1 and 2. Follicle cell differentiation appears to be independent of cell lineage, but is orchestrated by sequential cell interactions, and in particular by Notch signaling. Our results reveal the sophisticated, self-correcting behavior of an epithelial stem cell lineage at close to single-cell resolution.     

The Kinetics of Vitamin D3 in the Osteoblastic Cell

Experimental evidence is presented on the translocation of vitamin D metabolite, 1,25-(OH)₂D₃, from the membrane to the nucleus in osteoblast progenitor cells. A mathematical model permitting traversal of the cytoplasm at either a fixed velocity or by diffusion is formulated in order to determine whether transport along the cytoskeletal tracks is more consistent with the observed spatial-temporal distribution than diffusion, and it is so found. The model includes reactions in the nucleus involving D₃ to form other compounds, such as protegerin, and thus also makes predictions of the concentrations of these compounds in various regions of the cell.     

Probing the Interaction Forces of Prostate Cancer Cells with Collagen I and Bone Marrow Derived Stem Cells on the Single Cell Level

Adhesion of metastasizing prostate carcinoma cells was quantified for two carcinoma model cell lines LNCaP (lymph node-specific) and PC3 (bone marrow-specific). By time-lapse microscopy and force spectroscopy we found PC3 cells to preferentially adhere to bone marrow-derived mesenchymal stem cells (SCP1 cell line). Using atomic force microscopy (AFM) based force spectroscopy, the mechanical pattern of the adhesion to SCP1 cells was characterized for both prostate cancer cell lines and compared to a substrate consisting of pure collagen type I. PC3 cells dissipated more energy (27.6 aJ) during the forced de-adhesion AFM experiments and showed significantly more adhesive and stronger bonds compared to LNCaP cells (20.1 aJ). The characteristic signatures of the detachment force traces revealed that, in contrast to the LNCaP cells, PC3 cells seem to utilize their filopodia in addition to establish adhesive bonds. Taken together, our study clearly demonstrates that PC3 cells have a superior adhesive affinity to bone marrow mesenchymal stem cells, compared to LNCaP. Semi-quantitative PCR on both prostate carcinoma cell lines revealed the expression of two Col-I binding integrin receptors, α1β1 and α2β1 in PC3 cells, suggesting their possible involvement in the specific interaction to the substrates. Further understanding of the exact mechanisms behind this phenomenon might lead to optimized therapeutic applications targeting the metastatic behavior of certain prostate cancer cells towards bone tissue.     

The Molecular Profiles of Neural Stem Cell Niche in the Adult Subventricular Zone

Neural stem cells (NSCs) reside in a unique microenvironment called the neurogenic niche and generate functional new neurons. The neurogenic niche contains several distinct types of cells and interacts with the NSCs in the subventricular zone (SVZ) of the lateral ventricle. While several molecules produced by the niche cells have been identified to regulate adult neurogenesis, a systematic profiling of autocrine/paracrine signaling molecules in the neurogenic regions involved in maintenance, self-renewal, proliferation, and differentiation of NSCs has not been done. We took advantage of the genetic inducible fate mapping system (GIFM) and transgenic mice to isolate the SVZ niche cells including NSCs, transit-amplifying progenitors (TAPs), astrocytes, ependymal cells, and vascular endothelial cells. From the isolated cells and microdissected choroid plexus, we obtained the secretory molecule expression profiling (SMEP) of each cell type using the Signal Sequence Trap method. We identified a total of 151 genes encoding secretory or membrane proteins. In addition, we obtained the potential SMEP of NSCs using cDNA microarray technology. Through the combination of multiple screening approaches, we identified a number of candidate genes with a potential relevance for regulating the NSC behaviors, which provide new insight into the nature of neurogenic niche signals.     

Cell Lineage Identification and Stem Cell Culture in a Porcine Model for the Study of Intestinal Epithelial Regeneration

Significant advances in intestinal stem cell biology have been made in murine models; however, anatomical and physiological differences between mice and humans limit mice as a translational model for stem cell based research. The pig has been an effective translational model, and represents a candidate species to study intestinal epithelial stem cell (IESC) driven regeneration. The lack of validated reagents and epithelial culture methods is an obstacle to investigating IESC driven regeneration in a pig model. In this study, antibodies against Epithelial Adhesion Molecule 1 (EpCAM) and Villin marked cells of epithelial origin. Antibodies against Proliferative Cell Nuclear Antigen (PCNA), Minichromosome Maintenance Complex 2 (MCM2), Bromodeoxyuridine (BrdU) and phosphorylated Histone H3 (pH3) distinguished proliferating cells at various stages of the cell cycle. SOX9, localized to the stem/progenitor cells zone, while HOPX was restricted to the +4/‘reserve’ stem cell zone. Immunostaining also identified major differentiated lineages. Goblet cells were identified by Mucin 2 (MUC2); enteroendocrine cells by Chromogranin A (CGA), Gastrin and Somatostatin; and absorptive enterocytes by carbonic anhydrase II (CAII) and sucrase isomaltase (SIM). Transmission electron microscopy demonstrated morphologic and sub-cellular characteristics of stem cell and differentiated intestinal epithelial cell types. Quantitative PCR gene expression analysis enabled identification of stem/progenitor cells, post mitotic cell lineages, and important growth and differentiation pathways. Additionally, a method for long-term culture of porcine crypts was developed. Biomarker characterization and development of IESC culture in the porcine model represents a foundation for translational studies of IESC-driven regeneration of the intestinal epithelium in physiology and disease.