סך מח עצם הכנת <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> ארבעה עכברים B6 הם הקריבו גזור להשיג tibias, עצמות הירך, האגן ועמוד השדרה. </li><li style=";text-align:right;direction:rtl"> במהלך והניתוחים, הגפיים ועצמות נשמרים PBS 2% FCS מומת חום (אופציונלי 2mm EDA - בינוני דיסקציה). </li><li style=";text-align:right;direction:rtl"> העצמות הן נקיות כתוש עם מכתש ועלי במדיום לנתיחה. הדרך היעילה לעשות זאת היא לרסק tibias, עצמות הירך והירכיים של כל עכבר, אז 2 קוצים בכל פעם. </li><li style=";text-align:right;direction:rtl"> תערובת התא המתקבל העכבר כל הזמן נפרדים מסונן דרך מסנן 40μm לתוך צינור בז 50 מ&quot;ל. בדרך זו, כל שפופרת מכילה את התערובת תא שהושגו מתוך כל עצמות רגל של עכבר, וחצי של התערובת המתקבלת 2 קוצים. בשלב זה, המסנן משמש להפריד פסולת העצם. </li><li style=";text-align:right;direction:rtl"> זה בדרך כלל מומלץ לרסק את עצמות הרגל פעמיים כדי לקבל לבן (עם מח עצם לא), שברי עצמות, ואת שברי עמוד השדרה 2 או 3 פעמים. </li><li style=";text-align:right;direction:rtl"> ממלאים את צינורות יש להם מאוזנת, ספין 5 דקות 1200rpm. </li><li style=";text-align:right;direction:rtl"> לשאוב supernatant ו לשחרר את גלולה ידי גרירת צינורות על המדף צינור (שלב זה חשוב אחרי כל צנטריפוגה כדי למזער היווצרות גוש ואובדן התא). Resuspend כל הצינורית 1 מ&quot;ל PBS FCS 2% (NO EDTA מעתה והלאה) אם אתה עושה דלדול השושלת, או בכל אמצעי אחסון אחרת הנוחה לך. </li></ol> Lineage דלדול <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הליך זה מאפשר לך לחסל תאים מובחנים מאוד ולקבל אוכלוסייה הטרוגנית מאוד של תאים, המועשר בתאי גזע אב. אם אתה מיון HSC, צעד זה מפחית באופן דרסטי את מספר התאים כי צריך לעבור מיון התא, ולכן צמצום זמן מיון המאפשר לכם למיין HSC מתוך מספר גבוה יותר של עכברים, כך שתוכל להשיג מספר גבוה יותר של HSC במיון אחד. </li><li style=";text-align:right;direction:rtl"> אם אתה הולך למיין מאוחר יותר, להוציא 50μl תערובת תאים (אני בדרך כלל לוקח קצת מהצינור אחד) למיון שולטת (בלא כתם ו Sca1, c-Kit, CD48, Flk2 צבע שולט יחיד). </li><li style=";text-align:right;direction:rtl"> הוסף 30ml/tube לין קוקטייל. קוקטייל Lineage מכיל מגוון של נוגדנים, שלמרבה הצער שינויים קצת מן המעבדה למעבדה. במעבדה Scadden, אנו משתמשים נוגדנים נגד biotinilated Gr1, Ter119, CD4, CD8, CD3, B220. הדילול הסופי שלהם ב מכתים הוא 1:700. </li><li style=";text-align:right;direction:rtl"> וורטקס במהירות לערבב דגירה במשך 15 דקות ב 4 ° C. </li><li style=";text-align:right;direction:rtl"> במהלך הדגירה, להכין את העמודות על מגנטים וצינורות elution 15ml תחת העמודות. דגה PBS כמה באמצעות מסנן steriflip (אתה אמור לראות בועות אוויר קטנות נעות לעבר פני השטח של PBS בשל השאיפה ואקום), וכן לאזן את העמודות על ידי יישום PBS degassed 3ml כל עמודה. </li><li style=";text-align:right;direction:rtl"> זרימת טור הוא כ 1ml/5min, אז זה לוקח בערך 15 דקות כדי לקבל את העמודות מוכן לשימוש. </li><li style=";text-align:right;direction:rtl"> בסוף הדגירה, מוסיפים PBS 2% FCS כדי למלא את צינורות ספין למטה 5 דקות על 1200rpm. </li><li style=";text-align:right;direction:rtl"> לשאוב supernatant, לשחרר את כדורי ואת resuspend ב מ&quot;ל 1 / צינור degassed PBS. </li><li style=";text-align:right;direction:rtl"> אם אתה מיון מאוחר יותר, להוציא מח עצם הכולל Lineage אחד מכתים צבע ctrl (15 מ&quot;ל, קצת מהצינור כל אחד). </li><li style=";text-align:right;direction:rtl"> הוסף streptavidin חרוזים, 30ml/tube מערבולת במהירות לערבב דגירה במשך 15 דקות ב 4 ° C. </li><li style=";text-align:right;direction:rtl"> בסוף הדגירה, מוסיפים 2 מ&quot;ל / שפופרת של PBS degassed. שים צינורות אוסף חדש תחת העמודים, ולהחיל כל השעיה לעמודה, סינון דרך פילטר 40μm. </li><li style=";text-align:right;direction:rtl"> הוסף 3 מ&quot;ל של PBS degassed על צינור אחד לשטוף אותו. לאחר העמודות ריקות, חלה שוב באמצעות מסנן 40μm אותו. </li><li style=";text-align:right;direction:rtl"> Eluate מכיל השושלת מדולדל תא תערובת, אוכלוסייה הטרוגנית מועשר עבור ותאי גזע אבות. בריכת התוכן של 4 צינורות לתוך 2 צינורות. ספין דקות 5 ב 1500rpm. </li><li style=";text-align:right;direction:rtl"> לשאוב supernatant. גלולה צריך להיות לבן או לבן בעיקר. שחרר את כדורי ואת resuspend אותם יחד 1ml סך PBS FCS 2% אם אתה מיון מאוחר יותר, resuspend אחרת בכל נפח הנוחה לך. </li></ol> LKS או HSC מיון ארוך טווח <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> קח את השושלת מדולדל אחד מכתים צבע ctrl (15 מ&quot;ל). זה יאפשר לך להעריך את היעילות של דלדול השושלת שלך. </li><li style=";text-align:right;direction:rtl"> תערובת התא למיין הוא מוכתם בצינור 15 מ&quot;ל. </li></ol> הוספת נוגדנים: <table border="0" style=";text-align:right;direction:rtl"><tbody><tr><td> SCA PE-Cy5.5 </td><td> 1: 100 </td><td> 10 מ&quot;ל </td></tr><tr><td> ערכת APC </td><td> 1:100 </td><td> 10 מ&quot;ל </td></tr><tr><td> SA PE-Cy7 </td><td> 1:500 </td><td> 2ml </td></tr></tbody></table> כדי למיין HSC לטווח ארוך, גם להוסיף: <table border="0" style=";text-align:right;direction:rtl"><tbody><tr><td class="jove_content"> CD48 FITC </td><td class="jove_content"> 1:1000 </td><td class="jove_content"> 1ml </td></tr><tr><td class="jove_content"> Flk2 PE </td><td class="jove_content"> 1:200 </td><td class="jove_content"> 5 מ&quot;ל </td></tr&gt; </tbody></table> כמו כן, להכין מכתים אחת שולטת באמצעות מח עצם הכולל שמרו בצד לאחר דיסקציה לבין מח עצם הכולל מוכתם השושלת. Stainings אלה יכול להיעשות ישירות לתוך צינורות FACS. הקפד להיות בחושך! <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> וורטקס במהירות לערבב דגירה על 4 מעלות צלזיוס למשך 20 עד 30 דקות, ערבוב מחדש חצי דרך דרך. </li><li style=";text-align:right;direction:rtl"> כדי לשטוף את עודף של נוגדנים, למלא את צינורות עם PBS FCS 2% ספין דקות 5 ב 1500 סל&quot;ד. </li><li style=";text-align:right;direction:rtl"> הסר supernatant, resuspend כל כדורי ולהעביר אותם אל צינורות חדשים FACS עם כובעים הסינון. זה עלול לקחת זמן כדי לקבל את התאים לעבור את המסננים האלה, חשוב ליישם עוד קצת PBS לאחר מכן לשטוף את המסנן ולמזער את אובדן התא. צעד זה נחוץ כדי למנוע סתימת של סדרן, אשר הוא הסיוט הגרוע ביותר של כל משתמש סדרן התא. </li><li style=";text-align:right;direction:rtl"> כאשר חלוקת התאים למתקן מיון התא, הקפד גם להכין צינורית עם PBS 10% HI FCS שבו תאים ייאספו. </li><li style=";text-align:right;direction:rtl"> אם אתה מיון תאים LKS, תקבל אוכלוסיה הטרוגנית המכילה טווח ארוך טווח קצר repopulating אבות HSC ו multipotent. התשואה הממוצעת היא 300,000 LKS מ 4 עכברים. </li><li style=";text-align:right;direction:rtl"> אם השתמשת גם CD48 ואת Flk2 נוגדנים, ניתן להפריד בין האוכלוסייה של HSC לטווח ארוך, לטווח קצר HSC MPP. LT-HSC הם אוכלוסייה נדירה ביותר. התשואה הממוצעת היא כ -50,000 עד 60,000 תאים 4 עכברים. </li></ol>

isolation and transplantation of hematopoietic stem cells (hscs)

Watch this Scientific Journal Video about Isolation and Transplantation of Hematopoietic Stem Cells HSCs at JoVE.com

Isolation and Transplantation of Hematopoietic Stem Cells HSCs

בידוד והשתלות של בתאי גזע hematopoietic (HSCs)

isolation-and-transplantation-of-hematopoietic-stem-cells-hscs

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Biology

Isolation and Transplantation of Hematopoietic Stem Cells (HSCs)

Video: Isolation and Transplantation of Hematopoietic Stem Cells HSCs

Derivation of Hematopoietic Stem Cells from Murine Embryonic Stem Cells

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are derived from the blastocyst of the early embryo and are pluripotent in differentiative ability.  Their vast differentiative potential has made them the focus of much research centered on deducing how to coax them to generate clinically useful cell types.  The successful derivation of hematopoietic stem cells (HSC) from mouse ESC has recently been accomplished and can be visualized in this video protocol.  HSC, arguably the most clinically exploited cell population, are used to treat a myriad of hematopoietic malignancies and disorders.  However, many patients that might benefit from HSC therapy lack access to suitable donors.  ESC could provide an alternative source of HSC for these patients.  The following protocol establishes a baseline from which ESC-HSC can be studied and inform efforts to isolate HSC from human ESC.  In this protocol, ESC are differentiated as embryoid bodies (EBs) for 6 days in commercially available serum pre-screened for optimal hematopoietic differentiation.  EBs are then dissociated and infected with retroviral HoxB4.  Infected EB-derived cells are plated on OP9 stroma, a bone marrow stromal cell line derived from the calvaria of  M-CSF-/- mice, and co-cultured in the presence of hematopoiesis promoting cytokines for ten days.  During this co-culture, the infected cells expand greatly, resulting in the generation a heterogeneous pool of 100s of millions of cells.  These cells can then be used to rescue and reconstitute lethally irradiated mice.

This protocol details the derivation of transplantable hematopoietic stem cells from mouse embryonic stem cells (ESC) and their subsequent injection into lethally irradiated recipient mice. Briefly, ESC are differentiated as embryoid bodies, which are then infected with retroviral HoxB4 and co-cultured with OP9 stromal cells and hematopoietic cytokines.

גזירת בתאי גזע hematopoietic מ Murine בתאי גזע עובריים

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are ...

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. ...

Isolation of Early Hematopoietic Stem Cells from Murine Yolk Sac and AGM

In the mouse embryo, early hematopoiesis occurs simultaneously in multiple organs, which includes the yolk sac and aorta-gonad-mesonephros region. These regions are crucial in establishing the blood system in the embryos and leads to the eventual movement of stem cells into the fetal liver and then development of adult stem cells in the bonemarrow. Early hematopoietic stem cells can be isolated from these organs through microdissection of the embryo followed by flow cytometric sorting to obtain a more pure population. It remains unclear how these stem cell populations contribute to the fetal and adult stem cell pool.   Also, our lab investigates how early stem cells functionally differ from fetal and adult hematopoietic stem cells. Furthermore, our lab sorts different populations of hematopoietic stem cells and test their functional role in the context of a variety of genetic models. In this video, we demonstrate the micro-dissection procedure we commonly use and also show the results of a typical FACS plotfter isolating these rare populations, it is possible to perform a variety of functional assays including: colony assays and bone marrow transplants. 



This video shows how to micro-dissect the yolk sac and aorta-gonad-mesonephros region from embryos and use flow cytometry to sort hematopoietic stem cells.

בידוד מוקדם בתאי גזע hematopoietic מ Murine חלמון Sac ו AGM

In the mouse embryo, early hematopoiesis occurs simultaneously in multiple organs, which includes the yolk sac and aorta-gonad-mesonephros region. These ...

Generation of Human CD40-activated B cells

CD40-activated B cells (CD40-B cells) have been identified as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cancer immunotherapy 1-3. Compared to Dendritic cells (DCs), the best characterized APC, CD40-B cells have several distinct biological and technical properties. Similar to DCs, B cells show an increased expression of MHC and co-stimulatory molecules (Fig.1b), exhibit a strong migratory capacity and present antigen presentation efficiently to T cells, after stimulation with interleukin-4 and CD40 ligand (CD40L). However, in contrast to immature or mature DCs, CD40-B cells express the full lymph node homing triad consisting of CD62L, CCR7/CXCR4, and leukocyte function antigen-1 (LFA1, CD11a/CD18), necessary for homing to secondary lymphoid organs (Fig.1a) 3. CD40-B cells can be generated without difficulties from very small amounts of peripheral blood which can be further expanded in vitro to very large amounts of highly-pure CD40-B cells (&gt;109 cells per patient) from healthy donors as well as cancer patients (Fig.1c,d) 1,4. 
In this protocol we demonstrate how to obtain fully activated CD40-B cells from human PBMC. Key molecules for the cell culture are CD40 ligand, interleukin-4 (IL-4) and cyclosporin A (CsA), which are replenished in a 3-4 day culture cycle. For laboratory purposes CD40-stimulation is provided by NIH/3T3 cells expressing recombinant human CD40 ligand (tCD40L NIH/3T3) 5. To avoid contamination with non-transfected cells, expression of the human CD40 ligand on the transfectants has to be checked regularly (Fig.2). 
After 14 days CD40-B cell cultures consist of more than 95% pure B cells and an expansion of CD40-B cells over 65 days is frequently possible without any loss of function 1, 4. CD40-B cells efficiently take up, process and present antigens to T cells 6. They do not only prime na&#970;ve, but also expand memory T cells 7,8. CD40-activated B cells can be used to study B-cell activation, differentiation and function. Moreover, they represent a promising tool for therapeutic or preventive vaccination against tumors 9.

In this video we present the ex vivo generation and expansion of human CD40-activated B cells (CD40-B) from peripheral blood mononuclear cells (PBMC) by stimulation with CD40 ligand and interleukin-4.

הדור של האדם CD40 בתאי B המופעל

CD40-activated B cells (CD40-B cells) have been identified as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cancer ...

Isolation and Derivation of Mouse Embryonic Germinal Cells

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum (dpc) mouse embryos.  Developing gonadal ridges from mouse embryos (C57BL6J) were dissociated by mechanical disruption with collagenase, then plated in a mouse embryo fibroblast feeder layer (MEF-CF1) that was previously mitotically inactivated with mitomycin C in the presence of knockout media and supplemented with Leukemia Inhibitor Factor (LIF), basic Fibroblast Growth Factor (bFGF), and Stem Cell Factor (SCF).  Using these optimized methods for PCG identification, isolation, and establishment of culture conditions permits long term cultures of EG cells for more than 40 days.  The embryonic germinal cell lines showed embryonic phenotype and expression of common used markers of the pluripotent state.  Isolation and derivation of germinal cells in culture provide a tool to understand their development in vitro and offer the opportunity to monitor cumulative damage at genetic and epigenetic levels after exposure to oxidative stress.

The ability of embryonic germinal cells to differentiate into primordial germinal cells during early development stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum mouse embryos.

בידוד גזירת תאים עובריים בעכבר ז&#39;רמינל

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental ...

Lentiviral-mediated Knockdown During Ex Vivo Erythropoiesis of Human Hematopoietic Stem Cells

Erythropoiesis is a commonly used model system to study cell differentiation. During erythropoiesis, pluripotent adult human hematopoietic stem cells (HSCs) differentiate into oligopotent progenitors, committed precursors and mature red blood cells 1. This process is regulated for a large part at the level of gene expression, whereby specific transcription factors activate lineage-specific genes while concomitantly repressing genes that are specific to other cell types 2. Studies on transcription factors regulating erythropoiesis are often performed using human and murine cell lines that represent, to some extent, erythroid cells at given stages of differentiation 3-5. However transformed cell lines can only partially mimic erythroid cells and most importantly they do not allow one to comprehensibly study the dynamic changes that occur as cells progress through many stages towards their final erythroid fate. Therefore, a current challenge remains the development of a protocol to obtain relatively homogenous populations of primary HSCs and erythroid cells at various stages of differentiation in quantities that are sufficient to perform genomics and proteomics experiments.
Here we describe an ex vivo cell culture protocol to induce erythroid differentiation from human hematopoietic stem/progenitor cells that have been isolated from either cord blood, bone marrow, or adult peripheral blood mobilized with G-CSF (leukapheresis). This culture system, initially developed by the Douay laboratory 6, uses cytokines and co-culture on mesenchymal cells to mimic the bone marrow microenvironment. Using this ex vivo differentiation protocol, we observe a strong amplification of erythroid progenitors, an induction of differentiation exclusively towards the erythroid lineage and a complete maturation to the stage of enucleated red blood cells. Thus, this system provides an opportunity to study the molecular mechanism of transcriptional regulation as hematopoietic stem cells progress along the erythroid lineage. 
Studying erythropoiesis at the transcriptional level also requires the ability to over-express or knockdown specific factors in primary erythroid cells. For this purpose, we use a lentivirus-mediated gene delivery system that allows for the efficient infection of both dividing and non-dividing cells 7. Here we show that we are able to efficiently knockdown the transcription factor TAL1 in primary human erythroid cells. In addition, GFP expression demonstrates an efficiency of lentiviral infection close to 90%. Thus, our protocol provides a highly useful system for characterization of the regulatory network of transcription factors that control erythropoiesis.

Lentiviral-mediated Knockdown During Ex Vivo Erythropoiesis of Human Hematopoietic Stem Cells

An ex vivo protocol to generate mature human red blood cells from hematopoietic stem/progenitors is described. Additionally we describe an efficient lentiviral-delivery method to knockdown the transcription factor TAL1 in primary erythroid cells. The efficiency of lentivirus mediated gene delivery is demonstrated using GFP expressing viruses.

Lentiviral בתיווך מציאה במהלך Ex Vivo Erythropoiesis של אדם בתאי גזע hematopoietic

Erythropoiesis is a commonly used model system to study cell differentiation. During erythropoiesis, pluripotent adult human hematopoietic stem cells ...

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 ...

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

אינדוקציה וניתוח של אפיתל לmesenchymal מעבר

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

Isolation, Culture, and Transplantation of Muscle Satellite Cells

Muscle satellite cells are a stem cell population required for postnatal skeletal muscle development and regeneration, accounting for 2-5% of sublaminal nuclei in muscle fibers. In adult muscle, satellite cells are normally mitotically quiescent. Following injury, however, satellite cells initiate cellular proliferation to produce myoblasts, their progenies, to mediate the regeneration of muscle. Transplantation of satellite cell-derived myoblasts has been widely studied as a possible therapy for several regenerative diseases including muscular dystrophy, heart failure, and urological dysfunction. Myoblast transplantation into dystrophic skeletal muscle, infarcted heart, and dysfunctioning urinary ducts has shown that engrafted myoblasts can differentiate into muscle fibers in the host tissues and display partial functional improvement in these diseases. Therefore, the development of efficient purification methods of quiescent satellite cells from skeletal muscle, as well as the establishment of satellite cell-derived myoblast cultures and transplantation methods for myoblasts, are essential for understanding the molecular mechanisms behind satellite cell self-renewal, activation, and differentiation. Additionally, the development of cell-based therapies for muscular dystrophy and other regenerative diseases are also dependent upon these factors.

However, current prospective purification methods of quiescent satellite cells require the use of expensive fluorescence-activated cell sorting (FACS) machines. Here, we present a new method for the rapid, economical, and reliable purification of quiescent satellite cells from adult mouse skeletal muscle by enzymatic dissociation followed by magnetic-activated cell sorting (MACS). Following isolation of pure quiescent satellite cells, these cells can be cultured to obtain large numbers of myoblasts after several passages. These freshly isolated quiescent satellite cells or ex vivo expanded myoblasts can be transplanted into cardiotoxin (CTX)-induced regenerating mouse skeletal muscle to examine the contribution of donor-derived cells to regenerating muscle fibers, as well as to satellite cell compartments for the examination of self-renewal activities.

The isolation and culture of a pure population of quiescent satellite cells, a muscle stem cell population, is essential to the understanding of muscle stem cell biology and regeneration, as well as stem cell transplantation for therapies in muscular dystrophy and other degenerative diseases.&#160;

בידוד, תרבות והשתלה של תאי לווין שריר

Muscle satellite cells are a stem cell population required for postnatal skeletal muscle development and regeneration, accounting for 2-5% of sublaminal ...

Measurement of Mitochondrial Mass and Membrane Potential in Hematopoietic Stem Cells and T-cells by Flow Cytometry

A fine balance of quiescence, self-renewal, and differentiation is key to preserve the hematopoietic stem cell (HSC) pool and maintain lifelong production of all mature blood cells. In recent years cellular metabolism has emerged as a crucial regulator of HSC function and fate. We have previously demonstrated that modulation of mitochondrial metabolism influences HSC fate. Specifically, by chemically uncoupling the electron transport chain we were able to maintain HSC function in culture conditions that normally induce rapid differentiation. However, limiting HSC numbers often precludes the use of standard assays to measure HSC metabolism and therefore predict their function. Here, we report a simple flow cytometry assay that allows reliable measurement of mitochondrial membrane potential and mitochondrial mass in scarce cells such as HSCs. We discuss the isolation of HSCs from mouse bone marrow and measurement of mitochondrial mass and membrane potential post ex vivo culture. As an example, we show the modulation of these parameters in HSCs via treatment with a metabolic modulator. Moreover, we extend the application of this methodology on human peripheral blood-derived T cells and human tumor infiltrating lymphocytes (TILs), showing dramatic differences in their mitochondrial profiles, possibly reflecting different T cell functionality. We believe this assay can be employed in screenings to identify modulators of mitochondrial metabolism in various cell types in different contexts.

Here we describe a reliable method to measure mitochondrial mass and membrane potential in ex vivo cultured hematopoietic stem cells and T cells.

מדידת מיסת מיטוכונדריאלי ופוטנציאל ממברנה בתאי גזע ותאי T-תאים על ידי זרימה Cy,

A fine balance of quiescence, self-renewal, and differentiation is key to preserve the hematopoietic stem cell (HSC) pool and maintain lifelong production ...