JQW, SZ, SD and KC are supported by grant from the National Institutes of Health and the Staman Ogilvie Fund&#8212;Memorial Hermann Foundation.

<ol>
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The authors have nothing to disclose.

transcriptome יונקים היא מאוד מורכבת 34-38. טכנולוגית RNA-Seq משחקת תפקיד חשוב יותר ויותר במחקרים של ניתוח transcriptome, גילוי תמלילי רומן וגילוי הווריאציה נוקלאוטיד הבודד וכו &#39;יש לו יתרונות רבים על פני שיטות אחרות לניתוח ביטוי גנים. כפי שהוזכר ב...

תאי גזע hematopoietic הם תאי דם נדירים שמתגוררים בעיקר בנישה של מח העצם הבוגר. הם אחראים לייצור של תאים נדרשים כדי לחדש את הדם והמערכת החיסונית 1. כסוג של תאי גזע, HSCs מסוגל גם התחדשות עצמית והבחנה. הבהרת מנגנונים ששולטים בהחלטת גורלו של HSCs, כלפי שתי התחדשות עצמית או בידול, יציע הדרכה רבת ערך על המניפולציה של HSCs למחקר מחלת דם ושימוש קליני 2. אחת בעיות שעומדות בפני החוקרים היא שאפשר לקיים HSCs והרחיב במבחנה למידה מוגבלת מאוד; רובם המכריע של צאצאיהם נבדלים באופן חלקי בתרבות 2. על מנת לזהות רגולטורים מרכזיים השולטים על התהליכים של התחדשות עצמית והבחנה בקנה מידת הגנום, שנהגנו קו עכבר פרימיטיווי hematopoietic תא אב EML כמערכת מודל. ההוא שורת תאים נגזרה מ3,4 מח עצם העכברי. כאשר נמאס עם גורמי גדילה שונים, תאי EML יכולים להתמיין erythroid, מיאלואידית, ותאי הלימפה במבחנה 5. חשוב לציין, את שורות תאים יכולים להיות מופצים בכמות גדולה במדיום התרבות המכיל גורם תאי גזע (SCF) ועדיין שמירת multipotentiality. ניתן להפריד תאי EML לאוכלוסיות משנה של-SCA לין העצמי חידוש + CD34 + ומובחנים באופן חלקי תאי לין-SCA-CD34- המבוססים על סמני משטח CD34 וSCA 6. בדומה לטווח קצר HSCs, SCA + CD34 + תאים מסוגלים התחדשות עצמית. כאשר טופל בSCF, לין-SCA + תאי CD34 + במהירות יכול להתחדש אוכלוסייה מעורבת של לין-SCA + תאי CD34 + ולין-SCA-CD34- וממשיך להתרבות 6. שתי האוכלוסיות דומות במורפולוגיה ויש רמות דומות של mRNA c-kit וחלבון 6. תאי לין-SCA-CD34- מסוגלים הפצת באמצעי תקשורת המכילה IL-3 במקום SCF 3. Unveilinז הרגולטורים המרכזיים בהחלטת גורל תא EML תציע הבנה טובה יותר של מנגנונים תאיים ומולקולריים במעבר התפתחותי מוקדם במהלך hematopoiesis. על מנת לחקור את ההבדלים מולקולריים שבבסיס בין-SCA לין העצמי חידוש + CD34 + ותאי לין-SCA-CD34- מובחנים באופן חלקי, היינו RNA-Seq לזיהוי גנים הביעו באופן דיפרנציאלי. בפרט, אנו מתמקדים בגורמי שעתוק, כגורמי שעתוק הם קריטיים בקביעת גורל תא. RNA-Seq הוא גישה שפותחה לאחרונה שמנצלת את היכולות של רצף הדור הבא טכנולוגיות (NGS) לפרופיל ולכמת RNAs עיבד מהגנום 7,8. בקיצור, רנ&quot;א הכל הוא poly-נבחרים ומקוטע כתבנית template.The RNA הראשונית ולאחר מכן הוסבה לcDNA באמצעות טרנסקריפטאז ההפוך. על מנת למפות תעתיקי רנ&quot;א באורך מלא, באמצעות RNA שלם, שאינו מושפל לבניית ספריית cDNA הוא חשוב. לPURפוזה של רצף, רצפי מתאם ספציפיים מתווספים לשני הקצוות של cDNA. ואז, ברוב המקרים, מולקולות cDNA הם מוגברים על ידי PCR והרצף באופן תפוקה גבוהה. לאחר רצף, התוצאה כך קורא יכולה להיות מיושרת לגנום התייחסות ומסד נתוני transcriptome. מספר קורא את המפה לגן ההתייחסות נספרו, ומידע זה יכול לשמש כדי להעריך את רמת ביטוי הגן. קורא גם יכול להיות התאסף דה נובו ללא הגנום התייחסות, המאפשר המחקר של transcriptomes באורגניזמים שאינם מודל 9. טכנולוגית RNA-seq שימשה גם כדי לזהות isoforms אחוי 10-12, תמלילי רומן 13 ושילובי גן 14. בנוסף לזיהוי של גני המקודדים חלבונים, RNA-Seq יכול גם לשמש כדי לזהות רומן ולנתח ברמת שעתוק של RNA קידוד שאינו, כגון ארוך ללא קידוד 15,16 RNA, microRNA 17, siRNA וכו &#39;18. בגלל tהוא הדיוק של שיטה זו, זה נוצל לצורך זיהוי של וריאציות נוקלאוטיד בודדות 19,20. לפני הופעתו של טכנולוגיית RNA-Seq, microarray היה השיטה העיקרית המשמשת לניתוח פרופיל ביטוי גנים. בדיקות תוכנן מראש מסונתזות ולאחר מכן מחוברת למשטח מוצק ליצירת שקופיות microarray 21. mRNA מופק והמיר לcDNA. במהלך תהליך השעתוק לאחור, נוקלאוטידים שכותרתו fluorescently משולבים cDNA וcDNA יכול להיות הכלאה על שקופיות microarray. עוצמת האות שנאספו מנקודה מסוימת תלויה בכמות של cDNA מחייבת הבדיקה הספציפית באותו האזור 21. לעומת טכנולוגית RNA-Seq, יש microarray מספר מגבלות. ראשית, microarray מסתמך על הידע קיים מראש של ביאור גן, תוך טכנולוגית RNA-Seq היא מסוגלת לזהות תמלילי רומן ברמה היחסית גבוהה רקע, המגבילה את השימוש בו, כאשר GEרמת ביטוי ne היא נמוכה. חוץ מזה, יש לו את הטכנולוגיה RNA-Seq הרבה יותר גבוה טווח דינמי של זיהוי (8,000 לקפל) 7, ואילו, בשל רקע ורוויה של אותות, את הדיוק של microarray הוא מוגבל לשני הגנים הביעו מאוד ונח 7,22. לבסוף, בדיקות microarray שונות ביעילות ההכלאה שלהם, ההופכת את התוצאות פחות אמינות כאשר משווים רמות ביטוי היחסית של תעתיקים שונים בתוך דגימה אחת 23. למרות שיש RNA-Seq יתרונות רבים על פני microarray, ניתוח הנתונים שלה הוא מורכב. זה אחת הסיבות שחוקרים רבים עדיין משתמשים microarray במקום RNA-Seq. כלים ביואינפורמטיקה שונים הנדרשים לעיבוד נתונים RNA-Seq וניתוח 24. בין כמה רצפים של הדור הבא פלטפורמות (NGS), 454, Illumina, Torrent מוצק והיון הם אלה בשימוש נרחב ביותר. 454 היו פלטפורמת NGS המסחרית הראשונה. בניגוד לפלטפורמות רצף האחריםאורך כגון Illumina ומוצק, הפלטפורמה 454 מייצרת לקרוא יותר (בסיס 700 ממוצע כניסות) 25. עוד קורא טוב יותר לאפיון ראשוני של transcriptiome בשלהם גבוהים יותר להרכיב את יעילות 25. החסרון העיקרי של הפלטפורמה 454 הוא העלות הגבוהה שלה לmegabase של רצף. Illumina והפלטפורמות מוצקים לייצר קוראים עם מספרים מוגברים ואורכים קצרים. העלות לmegabase של רצף היא נמוכה בהרבה מהפלטפורמה 454. בשל המספר הגדול של קצר קורא לIllumina ופלטפורמות מוצקים, ניתוח נתונים הוא הרבה יותר אינטנסיבי המחשוב. מחירו של המכשיר וריאגנטים עבור סידור עבור פלטפורמת Ion Torrent הוא זול יותר וזמן רצף קצר יותר 25. עם זאת, שיעור השגיאה ואת העלות לmegabase של רצף נמצאים גבוהה יותר בהשוואה לIllumina ופלטפורמות מוצקה. יש פלטפורמות שונות יתרונות וחסרונות משל ודורשות שיטות שונות לניתוח נתונים. PLAיש לבחור tform המבוסס על מטרת הרצף ואת הזמינות של מימון. במאמר זה, אנחנו לוקחים את פלטפורמת Illumina RNA-Seq כדוגמא. אנחנו השתמשנו תא EML כמערכת מודל כדי לחקור את הרגולטורים המרכזיים בתא התחדשות עצמית EML ובידול, וסיפקנו שיטות מפורטות של בניית RNA-Seq ספרייה וניתוח נתונים לחישוב רמת ביטוי וגילוי תמליל רומן. שהראינו בפרסום הקודם שלנו שמחקר RNA-seq במערכת מודל EML 2, כאשר בשילוב עם בדיקה פונקציונלית (לדוגמא מציאה shRNA) מספק גישה חזקה בהבנת המנגנון המולקולרי של המוקדמים של בידול hematopoietic השלבים, ויכולים לשמש כ מודל לניתוח של תא התחדשות עצמית והבחנה באופן כללי.

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identification of key factors regulating self-renewal and differentiation in eml hematopoietic precursor cells by rna-sequencing analysis

תאי גזע hematopoietic (HSCs) משמשים קליניים לטיפול בהשתלה כדי לבנות מחדש את מערכת hematopoietic של מטופל במחלות רבות כגון לוקמיה ולימפומה. הבהרת מנגנוני שליטה חידוש עצמי HSCs ובידול חשוב ליישום של HSCs למחקר ושימושים קליניים. עם זאת, לא ניתן לקבל כמות גדולה של HSCs בשל חוסר היכולת שלהם להתרבות במבחנה. כדי להתגבר על מכשול זה, היינו קו עצם עכבר מח שמקורן בתאים, שורת תאי EML (erythroid, מיאלואידית, ולימפוציטית), כמערכת מודל למחקר זה. RNA-רצף (RNA-Seq) כבר בשימוש יותר ויותר כדי להחליף microarray ללימודי ביטוי גנים. אנו מדווחים כאן שיטה מפורטת של שימוש בטכנולוגית RNA-Seq כדי לחקור את גורמי מפתח הפוטנציאל ברגולציה של תא EML התחדשות עצמית והבחנה. הפרוטוקול הניתן במאמר זה מחולק לשלושה חלקים. הנקוב הראשוןלא מסביר כיצד תרבות תאי EML ונפרד לין-CD34 + ותאי לין-CD34-. החלק השני של הפרוטוקול מציע נהלים מפורטים להכנת RNA מוחלטת ובניית הספרייה העוקבת ברצף תפוקה גבוהה. החלק האחרון מתאר את השיטה לניתוח נתונים RNA-Seq ומסביר כיצד להשתמש בנתונים כדי לזהות גורמי שעתוק הביעו באופן דיפרנציאלי בין לין-CD34 + ותאי לין-CD34-. גורמי השעתוק באופן משמעותי ביותר באו לידי ביטוי באופן דיפרנציאלי זוהו להיות הרגולטורים המרכזיים פוטנציאל השליטה תא EML התחדשות עצמית והבחנה. בחלקו המסכם של מאמר זה, אנו מדגישים את השלבים העיקריים לביצוע מוצלח של ניסוי זה. לסיכום, מאמר זה מציע שיטה של ​​שימוש בטכנולוגית RNA-Seq לזהות רגולטורי פוטנציאל של התחדשות עצמית והבחנה בתאי EML. הגורמים המרכזיים שזוהו נתונים לאנליזה פונקציונלית במורד הזרם במבחנה ואניvivo n.

על מנת לנתח את גנים הביעו באופן דיפרנציאלי בלין-CD34 + ותאי לין-CD34- EML, אנו משתמשים בטכנולוגית RNA-Seq. איור 1 מציג את זרימת העבודה של הנהלים. לאחר הבידוד של תאים שליליים שושלת על ידי מיון תא מגנטי, נפרדנו לין-SCA + CD34 + ותאי לין-SCA-CD34- באמצעות FACS Aria. תאי EML מועשרים לין הוכת?...

Watch this Scientific Journal Video about Identification of Key Factors Regulating Self-renewal and Differentiation in EML Hematopoietic Precursor Cells by RNA-sequencing Analysis at JoVE.com

Identification of Key Factors Regulating Self-renewal and Differentiation in EML Hematopoietic Precursor Cells by RNA-sequencing Analysis

ניתוחי RNA-רצף וביואינפורמטיקה שמשו לזהות גורמי שעתוק הביעו באופן משמעותי ואופן דיפרנציאלי בתת אוכלוסיות לין-CD34 + ולין-CD34- של EMLcells עכבר. גורמי שעתוק אלו עשויים לשחק תפקיד חשוב בקביעה לעבור בין תאי לין-CD34- לין-CD34 + ומובחן באופן חלקי עצמי חידוש.

זיהוי של גורמי מפתח עם הרגולציה עצמית התחדשות והתמיינות בתאי EML המטופואטיות מבשר על ידי ניתוח RNA-רצף

Hematopoietic stem cells (HSCs) are used clinically for transplantation treatment to rebuild a patient&#39;s hematopoietic system in many diseases such as ...

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JoVE Journal

Biology

12.2K Views.  The University of Texas Graduate School of Biomedical Sciences at Houston.  ניתוחי RNA-רצף וביואינפורמטיקה שמשו לזהות גורמי שעתוק הביעו באופן משמעותי ואופן דיפרנציאלי בתת אוכלוסיות לין-CD34 + ולין-CD34- של EMLcells עכבר. גורמי שעתוק אלו עשויים לשחק תפקיד חשוב בקביעה לעבור בין תאי לין-CD34- לין-CD34 + ומובחן באופן חלקי עצמי חידוש. ...

Video: Identification of Key Factors Regulating Self-renewal and Differentiation in EML Hematopoietic Precursor Cells by RNA-sequencing Analysis

Isolation and Analysis of Hematopoietic Stem Cells from the Placenta

Hematopoietic stem cells (HSCs) have the ability to self-renew and generate all cell types of the blood lineages throughout the lifetime of an individual. All HSCs emerge during embryonic development, after which their pool size is maintained by self-renewing cell divisions. Identifying the anatomical origin of HSCs and the critical developmental events regulating the process of HSC development has been complicated as many anatomical sites participate during fetal hematopoiesis. Recently, we identified the placenta as a major hematopoietic organ where HSCs are generated and expanded in unique microenvironmental niches (Gekas, et al 2005, Rhodes, et al 2008). Consequently, the placenta is an important source of HSCs during their emergence and initial expansion.
In this article, we show dissection techniques for the isolation of murine placenta from E10.5 and E12.5 embryos, corresponding to the developmental stages of initiation of HSCs and the peak in the size of the HSC pool in the placenta, respectively. In addition, we present an optimized protocol for enzymatic and mechanical dissociation of placental tissue into single-cell suspension for use in flow cytometry or functional assays. We have found that use of collagenase for single-cell suspension of placenta gives sufficient yields of HSCs. An important factor affecting HSC yield from the placenta is the degree of mechanical dissociation prior to, and duration of, enzymatic treatment.
We also provide a protocol for the preparation of fixed-frozen placental tissue sections for the visualization of developing HSCs by immunohistochemistry in their precise cellular niches. As hematopoietic specific antigens are not preserved during preparation of paraffin embedded sections, we routinely use fixed frozen sections for localizing placental HSCs and progenitors.

We have identified the placenta as a major hematopoietic organ during development. We found that hematopoietic stem cells (HSCs) are both generated and expanded in the placenta in unique microenvironmental niches. Here, we describe experimental techniques required for isolation and visualization of HSCs in the mouse placenta.

בידוד ניתוח בתאי גזע hematopoietic מן השליה

Hematopoietic stem cells (HSCs) have the ability to self-renew and generate all cell types of the blood lineages throughout the lifetime of an individual. ...

The use of SC1 (Pluripotin) to Support mESC Self-renewal in the Absence of LIF

Mouse embryonic stem (ES) cells are conventionally cultured with Leukemia Inhibitory Factor (LIF) to maintain self-renewal.1 However, LIF is expensive and activation of the LIF/JAK/STAT3 pathway is not absolutely required to maintain the self-renewal state.2 The SC1 small molecule may be an economical alternative to LIF. SC1 functions through dual inhibition of Ras-GAP and ERK1.3 Illustration of its mechanism of action makes it a useful tool to study the fundamental molecular mechanism of self-renewal. Here we demonstrate the procedure for culturing mouse ES cells in the presence of SC1 and show that they are able to maintain self-renewal in the absence of LIF. Cells cultured with SC1 showed similar morphology compared to cells maintained with LIF. Both exhibited typical mouse ES morphology after five passages. Expression of typical pluripotency markers (Oct4, Sox2, Nanog, and SSEA1) was observed after five passages in the presence of SC1. Furthermore, SC1 caused no overt toxicity on mouse ES cells.

The use of SC1 Pluripotin to Support mESC Self-renewal in the Absence of LIF

SC1 functions through dual inhibition of Ras- GAP and ERK1. We tested the function of SC1 in supporting mouse ES cell self-renewal in the absence of LIF and showed that SC1 is able to maintain self-renewal of mouse ES cell cultures.

השימוש sc1 (Pluripotin) לתמיכה המסקלין עצמית חידוש בהעדר LIF

Mouse embryonic stem (ES) cells are conventionally cultured with Leukemia Inhibitory Factor (LIF) to maintain self-renewal.1 However, LIF is expensive and ...

Phenotypic Analysis and Isolation of Murine Hematopoietic Stem Cells and Lineage-committed Progenitors

The bone marrow is the principal site where HSCs and more mature blood cells lineage progenitors reside and differentiate in an adult organism. HSCs constitute a minute cell population of pluripotent cells capable of generating all blood cell lineages for a life-time1. The molecular dissection of HSCs homeostasis in the bone marrow has important implications in hematopoiesis, oncology and regenerative medicine. We describe the labeling protocol with fluorescent antibodies and the electronic gating procedure in flow cytometry to score hematopoietic progenitor subsets and HSCs distribution in individual mice (Fig. 1). In addition, we describe a method to extensively enrich hematopoietic progenitors as well as long-term (LT) and short term (ST) reconstituting HSCs from pooled bone marrow cell suspensions by magnetic enrichment of cells expressing c-Kit. The resulting cell preparation can be used to sort selected subsets for in vitro and in vivo functional studies (Fig. 2).
Both trabecular osteoblasts2,3 and sinusoidal endothelium4 constitute functional niches supporting HSCs in the bone marrow. Several mechanisms in the osteoblastic niche, including a subset of N-cadherin+ osteoblasts3 and interaction of the receptor tyrosine kinase Tie2 expressed in HSCs with its ligand angiopoietin-15 concur in determining HSCs quiescence. "Hibernation" in the bone marrow is crucial to protect HSCs from replication and eventual exhaustion upon excessive cycling activity6. Exogenous stimuli acting on cells of the innate immune system such as Toll-like receptor ligands7 and interferon-&alpha;6 can also induce proliferation and differentiation of HSCs into lineage committed progenitors. Recently, a population of dormant mouse HSCs within the lin- c-Kit+ Sca-1+ CD150+ CD48- CD34- population has been described8. Sorting of cells based on CD34 expression from the hematopoietic progenitors-enriched cell suspension as described here allows the isolation of both quiescent self-renewing LT-HSCs and ST-HSCs9. A similar procedure based on depletion of lineage positive cells and sorting of LT-HSC with CD48 and Flk2 antibodies has been previously described10. In the present report we provide a protocol for the phenotypic characterization and ex vivo cell cycle analysis of hematopoietic progenitors, which can be useful for monitoring hematopoiesis in different physiological and pathological conditions. Moreover, we describe a FACS sorting procedure for HSCs, which can be used to define factors and mechanisms regulating their self-renewal, expansion and differentiation in cell biology and signal transduction assays as well as for transplantation.

A method to analyse the distribution of bone marrow hematopoietic progenitors in flow cytometry as well as to efficiently isolate highly purified hematopoietic stem cells (HSCs) is described. The isolation procedure is essentially based on magnetic enrichment of c-Kit+ cells and cell sorting to purify HSCs for cellular and molecular studies.

ניתוח בידוד פנוטיפי של Murine בתאי גזע hematopoietic ו-Lineage מחויבים אבות

The bone marrow is the principal site where HSCs and more mature blood cells lineage progenitors reside and differentiate in an adult organism. HSCs ...

Oct4GiP Reporter Assay to Study Genes that Regulate Mouse Embryonic Stem Cell Maintenance and Self-renewal

Pluripotency and self-renewal are two defining characteristics of embryonic stem cells (ES cells). Understanding the underlying molecular mechanism will greatly facilitate the use of ES cells for developmental biology studies, disease modeling, drug discovery, and regenerative medicine (reviewed in 1,2).
To expedite the identification and characterization of novel regulators of ES cell maintenance and self-renewal, we developed a fluorescence reporter-based assay to quantitatively measure the self-renewal status in mouse ES cells using the Oct4GiP cells 3. The Oct4GiP cells express the green fluorescent protein (GFP) under the control of the Oct4 gene promoter region 4,5. Oct4 is required for ES cell self-renewal, and is highly expressed in ES cells and quickly down-regulated during differentiation 6,7. As a result, GFP expression and fluorescence in the reporter cells correlates faithfully with the ES cell identity 5, and fluorescence-activated cell sorting (FACS) analysis can be used to closely monitor the self-renewal status of the cells at the single cell level 3,8.
Coupled with RNAi, the Oct4GiP reporter assay can be used to quickly identify and study regulators of ES cell maintenance and self-renewal 3,8. Compared to other methods for assaying self-renewal, it is more convenient, sensitive, quantitative, and of lower cost. It can be carried out in 96- or 384-well plates for large-scale studies such as high-throughput screens or genetic epistasis analysis. Finally, by using other lineage-specific reporter ES cell lines, the assay we describe here can also be modified to study fate specification during ES cell differentiation.

We describe a fluorescence reporter assay to quickly identify and characterize genes that regulate mouse embryonic stem cell maintenance and self-renewal.

Oct4GiP Assay כתב ללמוד גנים המווסתים גזע עובריים עכבר נייד תחזוקה עצמית והתחדשות

Pluripotency and self-renewal are two defining characteristics of embryonic stem cells (ES cells). Understanding the underlying molecular mechanism will ...

In vivo Reprogramming of Adult Somatic Cells to Pluripotency by Overexpression of Yamanaka Factors

Induced pluripotent stem (iPS) cells that result from the reprogramming of somatic cells to a pluripotent state by forced expression of defined factors are offering new opportunities for regenerative medicine. Such clinical applications of iPS cells have been limited so far, mainly due to the poor efficiency of the existing reprogramming methodologies and the risk of the generated iPS cells to form tumors upon implantation.
We hypothesized that the reprogramming of somatic cells towards pluripotency could be achieved in vivo by gene transfer of reprogramming factors. In order to efficiently reprogram cells in vivo, high levels of the Yamanaka (OKSM) transcription factors need to be expressed at the target tissue. This can be achieved by using different viral or nonviral gene vectors depending on the target tissue. In this particular study, hydrodynamic tail-vein (HTV) injection of plasmid DNA was used to deliver the OKSM factors to mouse hepatocytes. This provided proof-of-evidence of in vivo reprogramming of adult, somatic cells towards a pluripotent state with high efficiency and fast kinetics. Furthermore no tumor or teratoma formation was observed in situ.
It can be concluded that reprogramming somatic cells in vivo may offer a potential approach to induce enhanced pluripotency rapidly, efficiently, and safely compared to in vitro performed protocols and can be applied to different tissue types in the future.

In vivo Reprogramming of Adult Somatic Cells to Pluripotency by Overexpression of Yamanaka Factors

This study demonstrates the reprogramming of somatic cells towards pluripotency in vivo without the generation of teratomas. We used hydrodynamic tail vein injection of plasmid DNA encoding the Yamanka factors to induce the in vivo reprogramming of adult hepatocytes into cells of enhanced pluripotency.

בתכנות מחדש vivo של תאים סומטיים למבוגרים לpluripotency ידי ביטוי יתר של גורמי יאמאנאקה

Induced pluripotent stem (iPS) cells that result from the reprogramming of somatic cells to a pluripotent state by forced expression of defined factors ...

Monitoring Functionality and Morphology of Vasculature Recruited by Factors Secreted by Fast-growing Tumor-generating Cells

The subcutaneous matrigel plug assay in mice is a method of choice for the in vivo evaluation of pro- and anti-angiogenic factors. In this method, desired factors are introduced into cold-liquid ECM-mimic gel which, after subcutaneous injection, solidifies to form an environment mimicking the cancer milieu. This matrix permits the penetration of host cells, such as endothelial cells, and therefore, the formation of vasculature.
Herein we propose a new modified matrigel plug assay, which can be exploited to illustrate the angiogenic potential of a pool of factors secreted by cancer cells, as opposed to a specific factor (e.g., bFGF and VEGF) or agent. The plug containing ECM-mimic gel is utilized to introduce the host (i.e., mouse) with a pool of factors secreted to the C.M. of fast-growing tumor-generating glioblastoma cells. We have previously described an extensive comparison of the angiogenic potential of U-87 MG human glioblastoma and its dormant-derived clone, in this system model, showing induced angiogenesis in the U-87 MG parental cells. The C.M. is prepared by filtering collected media from confluent tissue culture plates of either cell line following 48 hr incubation. Hence, it contains only factors secreted by the cells, without the cells themselves. Described here is the combination of two imaging modalities, microbubbles contrast-enhanced ultrasound imaging and intravital fibered-confocal endomicroscopy, for an accurate, real-time characterization of the extent, morphology and functionality of newly-formed blood vessels within the plugs.

We describe a matrigel plug assay to illustrate angiogenic potential of a pool of factors secreted by cancer cells using two complementary imaging modalities, ultrasound and endomicroscopy. The matrigel, an extracellular matrix (ECM)-mimic gel, is utilized to introduce the host (mouse) with angiogenic factors secreted to the conditioned media (C.M.).&#160;

פונקציונליות ניטור ומורפולוגיה של כלי דם גויס על ידי גורמים מופרשים על ידי תאים מניבי גידול בצמיחה מהיר

The subcutaneous matrigel plug assay in mice is a method of choice for the in vivo evaluation of pro- and anti-angiogenic factors. In this method, desired ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

במעקב 4 ממדי Vivo של תאי גזע ואבות היווצרות דם במבוגרי עכבר calvarial מח עצם

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Retroviral Infection of Murine Embryonic Stem Cell Derived Embryoid Body Cells for Analysis of Hematopoietic Differentiation

Embryonic stem cells (ESCs) are an outstanding model for elucidating the molecular mechanisms of cellular differentiation. They are especially useful for investigating the development of early hematopoietic progenitor cells (HPCs). Gene expression in ESCs can be manipulated by several techniques that allow the role for individual molecules in development to be determined. One difficulty is that expression of specific genes often has different phenotypic effects dependent on their temporal expression. This problem can be circumvented by the generation of ESCs that inducibly express a gene of interest using technology such as the doxycycline-inducible transgene system. However, generation of these inducible cell lines is costly and time consuming. Described here is a method for disaggregating ESC-derived embryoid bodies (EBs) into single cell suspensions, retrovirally infecting the cell suspensions, and then reforming the EBs by hanging drop. Downstream differentiation is then evaluated by flow cytometry. Using this protocol, it was demonstrated that exogenous expression of a microRNA gene at the beginning of ESC differentiation blocks HPC generation. However, when expressed in EB derived cells after nascent mesoderm is produced, the microRNA gene enhances hematopoietic differentiation. This method is useful for investigating the role of genes after specific germ layer tissue is derived.

Manipulating temporal gene expression in differentiating embryonic stem cells (ESCs) can be achieved using inducible gene systems. However, generation of these cell lines is costly and time consuming. This protocol achieves rapid expression of a transgene in differentiating ES-derived cells and subsequent analysis of downstream hematopoietic differentiation.

זיהום retroviral של Murine גזע עוברי תאי Embryoid גוף תא נגזר לניתוח של בידול המטופואטיות

Embryonic stem cells (ESCs) are an outstanding model for elucidating the molecular mechanisms of cellular differentiation. They are especially useful for ...

Fecal Glucocorticoid Analysis: Non-invasive Adrenal Monitoring in Equids

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, corticotrophin releasing hormone (CRH) is released from the hypothalamus in the brain. This stimulates the release of adrenocorticotrophic hormone (ACTH) from the pituitary gland, which in turn stimulates release of glucocorticoids from the adrenal gland. In horses the glucocorticoid corticosterone is responsible for several adaptations needed to support equine flight behaviour and subsequent removal from the aversive situation. Corticosterone metabolites can be detected in the feces of horses and assessment offers a non-invasive option to evaluate long term patterns of adrenal activity. Fecal assessment offers advantages over other techniques that monitor adrenal activity including blood plasma and saliva analysis. The non-invasive nature of the method avoids sampling stress which can confound results. It also allows the opportunity for repeated sampling over time and is ideal for studies in free ranging horses. This protocol describes the enzyme linked immunoassay (EIA) used to assess feces for corticosterone, in addition to the associated biochemical validation.

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. The method offers a non-invasive option to assess long term patterns in both domestic and free ranging horses. This protocol describes the enzyme linked immunoassay involved and the associated biochemical validation.

ניתוח Glucocorticoid צואה: לא פולשני ניטור הכליה ב Equids

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, ...

Analysis of Cell Suspensions Isolated from Solid Tissues by Spectral Flow Cytometry

Flow cytometry has been used for the past 40 years to define and analyze the phenotype of lymphoid and other hematopoietic cells. Initially restricted to the analysis of a few fluorochromes, currently there are dozens of different fluorescent dyes, and up to 14-18 different dyes can be combined at a time. However, several limitations still impair the analytical capabilities. Because of the multiplicity of fluorescent probes, data analysis has become increasingly complex due to the need of large, multi-parametric compensation matrices. Moreover, mutant mouse models carrying fluorescent proteins to detect and trace specific cell types in different tissues have become available, so the analysis (by flow cytometry) of auto-fluorescent cell suspensions obtained from solid organs is required. Spectral flow cytometry, which distinguishes the shapes of emission spectra along a wide range of continuous wavelengths, addresses some of these problems. The data is analyzed with an algorithm that replaces compensation matrices and treats auto-fluorescence as an independent parameter. Thus, spectral flow cytometry should be capable of discriminating fluorochromes with similar emission peaks and can provide a multi-parametric analysis without compensation requirements.
This protocol describes the spectral flow cytometry analysis, allowing for a 21-parameter (19 fluorescent probes) characterization and the management of an auto-fluorescent signal, providing high resolution in minor population detection. The results presented here show that spectral flow cytometry presents advantages in the analysis of cell populations from tissues difficult to characterize in conventional flow cytometry, such as the heart and the intestine. Spectral flow cytometry thus demonstrates the multi-parametric analytical capacity of high-performing conventional flow cytometry without the requirement for compensation and enables auto-fluorescence management.

This article describes spectral cytometry, a new approach in flow cytometry that uses the shapes of emission spectra to distinguish fluorochromes. An algorithm replaces compensations and can treat auto-fluorescence as an independent parameter. This new approach allows for the proper analysis of cells isolated from solid organs.

ניתוח של המתלים תא מבודד מרקמות Solid ידי זרימת cytometry ספקטרלי

Flow cytometry has been used for the past 40 years to define and analyze the phenotype of lymphoid and other hematopoietic cells. Initially restricted to ...