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

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The authors have nothing to disclose.

Transcriptome على الثدييات معقد جدا 34-38. التكنولوجيا-RNA يليها تلعب دورا متزايد الأهمية في دراسات تحليل Transcriptome على، كشف النصوص رواية واحدة النوكليوتيدات اكتشاف الاختلاف وما إلى ذلك لديه العديد من المزايا أكثر من غيرها من الأساليب لتحليل...

الخلايا الجذعية المكونة للدم هي خلايا الدم النادرة التي يقيمون بصورة رئيسية في نخاع عظم مكانة الكبار. أنها هي المسؤولة عن إنتاج الخلايا المطلوبة لتجديد الدم والجهاز المناعي 1. كنوع من الخلايا الجذعية، HSCs قادرون على حد سواء تجديد الذات والتمايز. توضيح الآليات التي تتحكم في قرار مصير HSCs، إما نحو التجديد الذاتي أو التمايز، سوف نقدم توجيهات قيمة بشأن التلاعب HSCs لأبحاث أمراض الدم والاستخدام السريري 2. واحد المشكلة التي يواجهها الباحثون هي أن HSCs يمكن الحفاظ على وتوسعت في المختبر على نطاق محدود جدا. ويفرق الغالبية العظمى من ذريتها جزئيا في الثقافة 2. من أجل تحديد الجهات التنظيمية الرئيسية التي تتحكم في عمليات تجديد الذات والتمايز على نطاق الجينوم واسعة، وكنا ماوس البدائية المكونة للدم خط خلية سلفية EML كنظام نموذج. عشروتم اشتقاق خط خلية من الفئران نخاع العظم 3،4. عندما تتغذى على عوامل النمو المختلفة، يمكن أن تفرق في خلايا EML محمر، النخاعي، والخلايا اللمفاوية في المختبر 5. الأهم من ذلك، يمكن نشر هذا خط الخلية بكميات كبيرة في الثقافة المتوسطة التي تحتوي على الخلايا الجذعية عامل (SCF) والإبقاء على تعدد الإمكانيات التي لا يزال. يمكن فصل الخلايا EML إلى مجموعات سكانية فرعية من تجديد الذات لين-SCA + CD34 + وتختلف جزئيا خلايا لين-SCA-CD34- على أساس علامات السطحية CD34 وSCA 6. على غرار المدى القصير HSCs، SCA + CD34 + الخلايا قادرة على تجديد الذات. عندما تعامل مع SCF، لين-SCA خلايا CD34 + + يمكن أن تتجدد بسرعة اختلط فيها لين-SCA خلايا CD34 + + ولين-SCA-CD34- وتستمر في التكاثر 6. السكان متشابهان في التشكل ولها مستويات مماثلة من مرنا ج-طقم 6 والبروتين. خلايا لين-SCA-CD34- قادرة على نشر في وسائل الإعلام التي تحتوي على IL-3 بدلا من SCF 3. Unveilinز المنظمين الرئيسيين في قرار مصير خلية EML سوف نقدم فهم أفضل للآليات الخلوية والجزيئية في التحول التنموي في وقت مبكر خلال الدم. من أجل التحقيق في الخلافات الجزيئية الكامنة بين تجديد الذات لين-SCA + CD34 + وخلايا متباينة جزئيا لين-SCA-CD34-، استخدمنا تسلسل الحمض النووي الريبي لتحديد الجينات وأعرب تفاضلي. على وجه الخصوص، ونحن نركز على عوامل النسخ، وعوامل النسخ حاسمة في تحديد مصير الخلية. تسلسل الحمض النووي الريبي هو نهج وضعت مؤخرا والتي تستخدم قدرات الجيل القادم التسلسل (خ ع) تقنيات للصفحة الشخصية وتحديد الرنا كتب من الجينوم 7،8. باختصار، هو الحمض النووي الريبي مجموع بولي-A المختارة ومجزأة كما هو ثم تحويل القالب template.The RNA الأولي في [كدنا] باستخدام الناسخ العكسي. من أجل تعيين النصوص الحمض النووي الريبي كامل طول، وذلك باستخدام سليمة، RNA غير المتدهورة لبناء مكتبة [كدنا] هو المهم. لبورتشكل التسلسل، يتم إضافة محول متواليات محددة لطرفي [كدنا]. بعد ذلك، في معظم الحالات، يتم تضخيم جزيئات [كدنا] بواسطة PCR والتسلسل بطريقة عالية الإنتاجية. بعد التسلسل، الناتجة يقرأ يمكن الانحياز إلى الجينوم مرجعية وقاعدة بيانات Transcriptome على. عدد يقرأ تلك الخريطة إلى الجين إشارة يحسب ويمكن استخدام هذه المعلومات لتقدير مستوى التعبير الجيني. ويمكن أيضا أن يتم تجميعها من جديد دون الجينوم إشارة، مما يتيح دراسة transcriptomes في الكائنات غير نموذج 9 يقرأ. كما تم استخدام تقنية الحمض النووي الريبي وما يليها لكشف الإسوية لصق 10-12، 13 رواية النصوص واندماج الجين 14. بالإضافة إلى الكشف عن الجينات المكودة للبروتين، يمكن أن تسلسل الحمض النووي الريبي أيضا أن تستخدم لكشف وتحليل مستوى الرواية نسخ من الرنا غير الترميز، مثل فترة طويلة غير الترميز الحمض النووي الريبي 15،16، 17 الرنا الميكروي، سيرنا الخ 18. بسبب رانه دقة هذه الطريقة، وقد استخدم ذلك للكشف عن الاختلافات النووية المنفردة 19،20. قبل ظهور التكنولوجيا-RNA بعدها، كان ميكروأري الوسيلة الرئيسية المستخدمة لتحليل التعبير الجيني الشخصي. ويتم تصنيعه تحقيقات المصممة مسبقا وتعلق بعد ذلك إلى سطح صلب لتشكيل شريحة ميكروأري 21. يتم استخراج مرنا وتحويلها إلى [كدنا]. أثناء عملية النسخ العكسي، يتم دمج النيوكليوتيدات fluorescently المسمى في [كدنا] ويمكن تهجين [كدنا] على الشرائح ميكروأري. شدة الإشارة التي تم جمعها من بقعة معينة تعتمد على كمية [كدنا] ملزم لجنة التحقيق بشكل خاص على تلك البقعة 21. بالمقارنة مع التكنولوجيا RNA يليها، ميكروأري لديه العديد من القيود. أولا، يعتمد ميكروأري على المعرفة الموجودة مسبقا من الشرح الجينات، في حين تكنولوجيا RNA بعدها قادرة على كشف النصوص رواية النسبي في مستوى الخلفية عالية، الأمر الذي يحد من استخدامها عند جنرال الكتريكمستوى التعبير شمال شرق منخفض. الى جانب ذلك، تكنولوجيا RNA يليها ديها أعلى بكثير من المدى الديناميكي الكشف (8000 مرات) 7، في حين، بسبب الخلفية وإشارات تشبع، دقة ميكروأري محدودة لكل من الجينات وأعرب عالية ومتواضع 7،22. أخيرا، تحقيقات ميكروأري تختلف في الكفاءة التهجين، والتي جعل نتائج أقل موثوقية عند مقارنة مستويات التعبير النسبية للمحاضر مختلفة في عينة واحدة 23. على الرغم من تسلسل الحمض النووي الريبي لديه العديد من المزايا أكثر من ميكروأري، وتحليل بياناتها معقد. وهذا هو أحد الأسباب أن العديد من الباحثين لا يزالون يستخدمون ميكروأري بدلا من تسلسل الحمض النووي الريبي. ويلزم أدوات المعلوماتية الحيوية المختلفة لمعالجة البيانات وتحليل الحمض النووي الريبي يليها 24. من بين العديد من الجيل المقبل من التسلسل (خ ع) المنصات، 454، البورشيد، الصلبة وايون سيل هم الأكثر استخداما على نطاق واسع. وكان 454 أول منصة تجارية NGS. خلافا لغيرها من المنصات التسلسلطول مثل البورشيد ومتين، ويولد 454 منصة تعد قراءة (متوسط ​​700 قاعدة يقرأ) 25. يعد يقرأ بشكل أفضل لتوصيف الأولي transcriptiome ترجع الزيادة إلى ارتفاع كفاءة تجميع 25. العيب الرئيسي للمنصة 454 هو التكلفة العالية في megabase التسلسل. البورشيد والمنصات الصلبة توليد يقرأ مع زيادة أعداد وأطوال قصيرة. التكلفة لكل megabase من تسلسل هو أقل بكثير من 454 منصة. بسبب الأعداد الكبيرة من يقرأ قصيرة لالبورشيد والمنصات الصلبة، تحليل بيانات أكثر كثافة حسابيا من ذلك بكثير. سعر الصك والكواشف لتسلسل لمنصة ايون سيل أرخص ووقت أقصر تسلسل 25. ومع ذلك، فإن نسبة الخطأ والتكلفة لكل megabase من تسلسل وأعلى بالمقارنة مع البورشيد والمنصات الصلبة. المنصات المختلفة لها مزاياها وعيوبها، وتتطلب أساليب مختلفة لتحليل البيانات. جيش التحرير الشعبى الصينىtform يجب أن يتم اختياره على أساس التسلسل الغرض وتوافر التمويل. في هذه الورقة، ونحن نأخذ منصة البورشيد RNA تسلسل كمثال. كنا خلية EML كنظام نموذج للتحقيق المنظمين الرئيسيين في EML خلية تجديد الذات والتمايز، وفرت طرق مفصلة ل-RNA يليها بناء مكتبة وتحليل البيانات لحساب مستوى التعبير والكشف عن نسخة الرواية. أظهرنا في نشر رسائلنا السابقة أن الدراسة وما يليها-RNA في EML نظام نموذجي 2، عندما يقترن اختبار وظيفي (على سبيل المثال shRNA ضربة قاضية) توفير نهج قوي في فهم الآلية الجزيئية للمراحل المبكرة من التمايز للدم، ويمكن أن تكون بمثابة نموذج لتحليل خلية تجديد الذات والتمايز بشكل عام.

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

وتستخدم الخلايا الجذعية المكونة للدم (HSCs) سريريا للعلاج زرع لإعادة بناء نظام المكونة للدم المريض في العديد من الأمراض مثل سرطان الدم وسرطان الغدد الليمفاوية. توضيح آليات السيطرة HSCs تجديد الذات والتمايز المهم للتطبيق HSCs للبحوث واستخدامات السريرية. ومع ذلك، فإنه من غير الممكن الحصول على كمية كبيرة من HSCs بسبب عدم قدرتها على التكاثر في المختبر. للتغلب على هذه العقبة، استخدمنا خط خلية نخاع المستمدة الماوس العظام، وقائمة الأدوية الأساسية (محمر، متعلق بالنخاع الشوكي، والليمفاوي) خط الخلية، كنظام نموذج لهذه الدراسة. RNA-التسلسل (RNA تسلسل) قد استخدمت على نحو متزايد ليحل محل ميكروأري للدراسات التعبير الجيني. نحن هنا تقرير مفصل لطريقة استخدام التكنولوجيا-RNA بعدها للتحقيق في العوامل الرئيسية المحتملة في تنظيم خلية EML تجديد الذات والتمايز. وينقسم البروتوكول المقدمة في هذه الورقة إلى ثلاثة أجزاء. على قدم المساواة الأولر يشرح كيفية الثقافة خلايا EML، وفصل لين-CD34 + والخلايا لين CD34-. الجزء الثاني من البروتوكول يوفر إجراءات مفصلة لمجموع RNA إعداد وبناء مكتبة لاحق الإنتاجية العالية التسلسل. ويصف الجزء الأخير طريقة لتحليل البيانات RNA وبعدها يشرح كيفية استخدام البيانات لتحديد عوامل النسخ أعرب تفاضلي بين لين-CD34 + والخلايا لين CD34-. تم تحديد عوامل النسخ الأهم أعرب تفاضلي أن المنظمين الرئيسيين المحتملين السيطرة على خلية EML تجديد الذات والتمايز. في قسم مناقشة هذه الورقة، ونحن تسليط الضوء على الخطوات الرئيسية للأداء الناجح لهذه التجربة. وباختصار، فإن هذه الورقة تقدم طريقة استخدام التكنولوجيا لتحديد تسلسل الحمض النووي الريبي التنظيمية المحتملة لتجديد الذات والتمايز في الخلايا EML. تتعرض العوامل الرئيسية المحددة لتحليل وظيفي المصب في المختبر وأنان الجسم الحي.

من أجل تحليل الجينات وأعرب تفاضلي في لين-CD34 + والخلايا لين CD34- EML، استخدمنا التكنولوجيا-RNA يليها. ويظهر الشكل 1 سير الإجراءات. بعد عزل الخلايا النسب السلبية من قبل الفرز خلية المغناطيسي، وانفصلنا لين-SCA CD34 + + والخلايا لين-SCA-CD34- باستخدام نظام مراقبة الأصول الميد?...

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

استخدمت RNA-التسلسل والتحليلات المعلوماتية الحيوية لتحديد عوامل النسخ أعرب كبير وبشكل مختلف في لين-CD34 + ولين CD34- القطعان من EMLcells الماوس. هذه عوامل النسخ قد تلعب أدوارا مهمة في تحديد التبديل بين الخلايا لين لين CD34--CD34 + ومتباينة جزئيا تجديد الذات.

تحديد العوامل الرئيسية تنظيم-تجديد الذات والتمايز في الخلايا المكونة للدم EML السلائف بواسطة تحليل الحمض النووي الريبي التسلسل

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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12.2K Views.  The University of Texas Graduate School of Biomedical Sciences at Houston.  استخدمت RNA-التسلسل والتحليلات المعلوماتية الحيوية لتحديد عوامل النسخ أعرب كبير وبشكل مختلف في لين-CD34 + ولين CD34- القطعان من EMLcells الماوس. هذه عوامل النسخ قد تلعب أدوارا مهمة في تحديد التبديل بين الخلايا لين لين CD34--CD34 + ومتباينة جزئيا تجديد الذ...

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.

العزلة وتحليل الخلايا الجذعية المكونة للدم من المشيمة

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.

استخدام SC1 (Pluripotin) لدعم MESC التجديد الذاتي في غياب 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.

المظهري التحليل وعزل الخلايا الجذعية المكونة للدم الفئران والأسلاف النسب، الملتزم

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 الفحص مراسل لدراسة الجينات التي تنظم الفأر الخلايا الجذعية الجنينية الصيانة والتجديد الذاتي

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.

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;

مراقبة وظائف والصرف من الأوعية الدموية تجنيده من قبل العوامل التي يفرزها الخلايا المولدة للورم النمو السريع

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.

في فيفو تتبع 4 الأبعاد للالجذعية المكونة للدم والسلف الكبار في خلايا الفأر قبي نخاع العظم

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.

عدوى فيروسات الفئران الجنينية الجذعية المستمدة خلية خلايا الجسم مضغي لتحليل التمايز لدم

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.

برازي تحليل جلايكورتيكود: رصد الغدة الكظرية غير الغازية في الخيليات

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.

تحليل معلقات خلية معزولة من الأنسجة الصلبة من قبل الطيفي التدفق الخلوي

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 ...