העבודה נתמכה על ידי מענקי Helse ובכפוף Rehabilitering (JLB), נורווגית המועצה למחקר (GH ו JCG) של אוניברסיטת אוסלו (NK ו JCG).

<ol>
	<li>Wichterle, H., Lieberam, I., Porter, J. A., Jessell, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directed+differentiation+of+embryonic+stem+cells+into+motor+neurons.">Directed differentiation of embryonic stem cells into motor neurons.</a> Cell. 110, 385-397 (2002).</li><li>Sigurjonsson, O. E., Perreault, M. C., Egeland, T., Glover, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adult+human+hematopoietic+stem+cells+produce+neurons+efficiently+in+the+regenerating+chicken+embryo+spinal.">Adult human hematopoietic stem cells produce neurons efficiently in the regenerating chicken embryo spinal.</a> Proc. Natl. Acad. Sci. U.S.A. 102, 5227-5232 (2005).</li><li>Pochampally, R. R., Neville, B. T., Schwarz, E. J., Li, M. M., Prockop, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rat+adult+stem+cells+(marrow+stromal+cells)+engraft+and+differentiate+in+chick+embryos+without+evidence+of+cell+fusion.">Rat adult stem cells (marrow stromal cells) engraft and differentiate in chick embryos without evidence of cell fusion.</a> Proc. Natl. Acad. Sci. U.S.A. 101, 9282-9285 (2004).</li><li>Goldstein, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transplantation+of+human+embryonic+stem+cells+to+the+chick+embryo.">Transplantation of human embryonic stem cells to the chick embryo.</a> Methods Mol. Biol. 331, 137-151 (2006).</li><li>Lee, H., Shamy, G. A., Elkabetz, Y., Schofield, C. M., Harrsion, N. L., Panagiotakos, G., Socci, N. D., Tabar, V., Studer, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directed+differentiation+and+transplantation+of+human+embryonic+stem+cell-derived+motoneurons.">Directed differentiation and transplantation of human embryonic stem cell-derived motoneurons.</a> Stem Cells. 25, 1931-1939 (2007).</li><li>Wichterle, H., Peljto, M., Nedelec, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Xenotransplantation+of+embryonic+stem+cell-derived+motor+neurons+into+the+developing+chick+spinal+cord.">Xenotransplantation of embryonic stem cell-derived motor neurons into the developing chick spinal cord.</a> Methods Mol. Biol. 482, 171-183 (2009).</li><li>Westerlund, U., Svensson, M., Moe, M. C., Varghese, M., Gustavsson, B., Wallstedt, L., Berg-Johnsen, J., Langmoen, I. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endoscopically+harvested+stem+cells:+a+putative+method+in+future+autotransplantation.">Endoscopically harvested stem cells: a putative method in future autotransplantation.</a> Neurosurgery. 57, 779-784 (2005).</li><li>Boquest, A. C., Shahdadfar, A., Brinchmann, J. E., Collas, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+stromal+stem+cells+from+human+adipose+tissue.">Isolation of stromal stem cells from human adipose tissue.</a> Methods Mol. Biol. 325, 35-46 (2006).</li><li>Smeland, E. B., Funderud, S., Kvalheim, G., Gaudernack, G., Rasmussen, A. M., Rusten, L., Wang, M. Y., Tindle, R. W., Blomhoff, H. K., Egeland, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+characterization+of+human+hematopoietic+progenitor+cells:+an+effective+method+for+positive+selection+of+CD34++cells.">Isolation and characterization of human hematopoietic progenitor cells: an effective method for positive selection of CD34+ cells.</a> Leukemia. 6, 845-852 (1992).</li><li>Zhang, X. Y., La Russa, V. F., Bao, L., Kolls, J., Schwarzenberger, P., Reiser, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lentiviral+vectors+for+sustained+transgene+expression+in+human+bone+marrow-derived+stromal+cells.">Lentiviral vectors for sustained transgene expression in human bone marrow-derived stromal cells.</a> Mol. Ther. 5, 555-565 (2002).</li><li>Hamburger, V., Hamilton, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+series+of+normal+stages+in+the+development+of+the+chick+embryo.">A series of normal stages in the development of the chick embryo.</a> J. Morph. 88 (1), 44-92 (1951).</li><li>Glover, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retrograde+and+anterograde+axonal+tracing+with+fluorescent+dextrans+in+the+embryonic+nervous+system.">Retrograde and anterograde axonal tracing with fluorescent dextrans in the embryonic nervous system.</a> Neuroscience Protocols. 30, 1-13 (1995).</li><li>O'Donovan, M. J., Bonnot, A., Mentis, G. Z., Arai, Y., Chub, N., Shneider, N. A., Wenner, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+the+spatiotemporal+organization+of+neural+activity+in+the+developing+spinal.">Imaging the spatiotemporal organization of neural activity in the developing spinal.</a> Dev. Neurobiol. 68, 788-803 (2008).</li><li>Mendelson, B., Frank, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specific+monosynaptic+sensory-motor+connections+form+in+the+absence+of+patterned+neural+activity+and+motoneuronal+cell+death.">Specific monosynaptic sensory-motor connections form in the absence of patterned neural activity and motoneuronal cell death.</a> J. Neurosci. 11, 1390-1403 (1991).</li><li>Glover, J. C., Boulland, J. L., Halasi, G., Kasumacic, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chimeric+Animal+Models+in+Human+Stem+Cell+Biology.">Chimeric Animal Models in Human Stem Cell Biology.</a> ILAR Journal. 51, 62-73 (2009).</li><li>Lee, G., Kim, H., Elkabetz, Y., Al Shamy, G., Panagiotakos, G., Barberi, T., Tabar, V., Studer, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+directed+differentiation+of+neural+crest+stem+cells+derived+from+human+embryonic+stem+cells.">Isolation and directed differentiation of neural crest stem cells derived from human embryonic stem cells.</a> Nat. Biotechnol. 25, 1468-1475 (2007).</li><li>Jiang, X., Gwye, Y., McKeown, S. J., Bronner-Fraser, M., Lutzko, C., Lawlor, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+characterization+of+neural+crest+cells+derived+from+in+vitro+differentiated+human+embryonic+stem+cells.">Isolation and characterization of neural crest cells derived from in vitro differentiated human embryonic stem cells.</a> Stem Cells Dev. 18, 1059-1070 (2008).</li></ol>

אין ניגודי אינטרסים הכריז.

עובר התרנגולת הוא מודל חיה נוח ומגוון ניסויים xenotransplantation. העדר מערכת חיסונית תפקודית במהלך ההתפתחות העוברית ואת העובר מאפשר הישרדות התפתחותם של תאים ממינים כל כך סובל את טמפרטורת הדגירה. עוברי עוף שימשו כדי לבדוק את פוטנציאל ההתחדשות של סוגים שונים של בתאי גזע (הנסקרת נ&quot;צ. 15). בתאי גזע היונקים יכולים להשתלב ברקמות העובר עוף כולל מערכת העצבים המרכזית, שם הם יכולים להצמיח נוירונים אשר התחזיות axonal, קישוריות הסינפטי ומאפיינים אלקטרו ניתן להעריך בהקשר של 1,2 מעגלים עצביים פונקציונלי. מכיוון ניתוחים קלים יחסית, מבוצעים תחת זריקות חזותי במקום לשלוט stereotaxic, מספר העוברים יכול בקלות להיות מוגברת כדי להכיל תנאים ניסויים שונים בתוך ניסוי יחיד. בדרך כלל התאים יהיה ניתן להזריק לתוך 20-30 עוברים בתוך הניסוי 4 שעות. תמותה לאחר הזרקה של עוברים הוא הגורם המגביל העיקרי. אמצעי זהירות יש לנקוט כדי למנוע דימום והתייבשות. שימו לב כי ישנם מסוימים בשלבי התפתחות קריטית שבה שיעור התמותה עולה טבעי. להשלים את העובר עם מ&quot;ל כמה הזרקה חם, עיקור עוף הצלצול הבא יום לפני שלבי התפתחות קריטיים, כמו גם להבטיח את החלון הביצה היטב אטום מכוונת כדי למנוע דליפה, משפר הישרדות. אם יש דליפה דרך אטם את הקלטת אמורה להתרחש, להסיר את הסרט, לייבש את המשטחים מחדש הקלטת. חוקרים אחדים להשתמש שעווה זכוכית או פלסטיק coverslips לאטום את החלון. התמותה לא צריך להיות שונה באופן משמעותי לעומת הזריק דמה המופעלים עוברים. למרות שאנו מתמקדים כאן על זריקה של בתאי גזע אנושיים לתוך anlage של המוח וחוט השדרה, בתאי גזע יכול גם להיות מוזרק anlage של איברים אחרים (ראו נ&quot;צ למשל. 4). כל איבר מציבה אתגרים משלה. לדוגמה, הזרקה לתוך הלב יכול להיות מאתגר בגלל התנגדות מכנית גבוהה פיפטה חדירה ובגלל התנועות, אשר מצד שני יכול להיות האטה על ידי קירור עוברים לטמפרטורת החדר לפני ההזרקה. איברים כי חוסר לומן לא יכול להכיל את הזרקת כמויות מספיקות ולכן עשויים לחייב פגיעה כירורגית כדי ליצור כלי קיבול. מניסיוננו, לאחר הנגע התחדשות רקמות יתרון משום שהוא מקדם את שילובם של בתאי גזע אנושיים לתוך הרקמה. תאים יכולים גם להיות מוזרק mesenchyme עובריים כגון mesenchyme somitic פיזור או הרכס העצבי נודדים כדרך להשיג הכללתו נגזרים mesenchymal, כולל הגרעינים לפסגה הנגזרות עצביים היקפיים 16,17.

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xenotransplantation of human stem cells into the chicken embryo

עובר התרנגולת הוא מודל חיה קלאסית לחקר ההתפתחות העוברית של העובר תקין לניסויים xenotransplantation ללמוד את התנהגותם של תאים סטנדרטית<em&gt; In vivo</em&gt; איכות הסביבה. היתרונות העיקריים של העובר עוף כוללים עלות נמוכה, נגישות גבוהה, קלות מניפולציה כירורגית היעדר מערכת חיסונית מפותחת. Xenotransplantation לעוברי עוף יכול לספק מידע רב ערך על התפשטות תאים, התמיינות התנהגות, התגובות של התאים אותות מוגדר נישות רקמה עוברית, ואת הפוטנציאל tumorigenic. השתלת התאים לעוברי עוף יכול להיות גם צעד לקראת ניסויים השתלת במודלים של בעלי חיים אחרים. לאחרונה העובר עוף נעשה שימוש כדי להעריך את הפוטנציאל neurogenic של גזע האדם ובתאים בעקבות ההשתלה לתוך anlage עצביים<sup&gt; 1-6</sup&gt;. בסרטון הזה אנחנו המסמך ההליך כולו להשתלה בתאי גזע אנושיים למערכת העצבים המרכזית בפיתוח של העובר עוף. ההליך מתחיל עם הדגירה של ביציות מופרות עד עוברים בגיל הרצוי פיתחו. קליפת נפתח אז, בניגוד העובר על ידי הזרקת צבע בין העובר לבין החלמון. נגעים קטנים עשויים בצינור העצבי באמצעות microsurgery, יצירת אתר משובי להפקדה תא המקדמת אינטגרציה הבאים לרקמת המארח. אנו מדגימים זריקות בתאי גזע אנושיים לתוך נגעים כאלה שנעשו בחלק של הצינור העצבי המהווה למוח האחורי ואת חוט השדרה, אל לומן החלק של הצינור העצבי שיוצר המוח. זריקות מערכתית לתוך הוורידים והעורקים extraembryonic הם הפגינו גם כדרך חלופית, כדי לספק תאים לרקמות vascularized כולל מערכת העצבים המרכזית. לבסוף אנו מראים כיצד להסיר את העובר מן הביצה לאחר כמה ימים של התפתחות נוספת ואיך לנתח את חוט השדרה חינם עבור ניתוחים פיזיולוגיים, היסטולוגית או ביוכימי הבאים.

Watch this Scientific Journal Video about Xenotransplantation of Human Stem Cells into the Chicken Embryo at JoVE.com

Xenotransplantation of Human Stem Cells into the Chicken Embryo

במאמר זה אנו מציגים שיטה השתלת בתאי גזע האדם לאזורים שונים של מערכת העצבים המרכזית של העובר עוף. זה מספק<em&gt; In vivo</em&gt; מודל להערכת התפשטות והבחנה של סוגים שונים של בתאי גזע אנושיים בסביבות הרקמה העוברית.

Xenotransplantation בתאי גזע אנושיים לתוך העובר עוף

The chicken embryo is a classical animal model for studying normal embryonic and fetal development and for xenotransplantation experiments to study the ...

xenotransplantation-of-human-stem-cells-into-the-chicken-embryo

Research

JoVE Journal

Biology

19.1K Views.  University of Oslo. במאמר זה אנו מציגים שיטה השתלת בתאי גזע האדם לאזורים שונים של מערכת העצבים המרכזית של העובר עוף. זה מספק In vivo מודל להערכת התפשטות והבחנה של סוגים שונים של בתאי גזע אנושיים בסביבות הרקמה העוברית.

Video: Xenotransplantation of Human Stem Cells into the Chicken Embryo

Transfecting Human Neural Stem Cells with the Amaxa Nucleofector

Transfection of primary mammalian neural cells, such as human neural stem/precursor cells (hNSPCs), with commonly used cationic lipid transfection reagents has often resulted in poor cell viability and low transfection efficiency.  Other mechanical methods of introducing a gene of interest, such as a &#8220;gene gun&#8221; or microinjection, are also limited by poor cell viability and low numbers of transfected cells.  The strategy of using viral constructs to introduce an exogenous gene into primary cells has been constrained by both the amount of time and labor required to create viral vectors and potential safety concerns.  We describe here a step-by-step protocol for transfecting hNSPCs using Amaxa's Nucleofector device and technology with electrical current parameters and buffer solutions specifically optimized for transfecting neural stem cells.  Using this protocol, we have achieved initial transfection efficiencies of ~35% and ~70% after stable transfection.  The protocol entails combining a high number of hNSPCs with the DNA to be transfected in the appropriate buffer followed by electroporation with the Nucleofector device.

Introducing a gene of interest into a cell is a powerful method for elucidating its function in vivo. This protocol describes an efficient method of transfecting a culture of human neural stem/precursor cells (hNSPCs) using the Nucleofector electroporation apparatus made by Amaxa.

Transfecting אדם בתאי גזע עצביים עם Nucleofector Amaxa

Transfection of primary mammalian neural cells, such as human neural stem/precursor cells (hNSPCs), with commonly used cationic lipid transfection ...

Counting Human Neural Stem Cells

Knowledge of the exact number of viable cells in a given volume of a cell suspension is required for many routine tissue culture manipulations, such as plating cells for immunocytochemistry or for cell transfections. This protocol describes a straightforward and fast method for differentiating between live and dead cells and quantifying the cell concentration and total cell number using a hemacytometer. This procedure first requires detaching cells from a growth surface and resuspending them in media. Next, the cells are diluted in a solution of Trypan blue (ideally to a concentration that will give 20-50 cells per quadrant) and placed in the hemacytometer. Finally, averaging the counts of viable cells in several randomly selected quadrants, dividing the average by the volume of one 1 mm2 quadrant (0.1 &mu;l) and multiplying by the dilution factor gives the number of cells per l. Multiplying this cell concentration by the total volume in &mu;l gives the total cell number. This protocol describes counting human neural stem/precursor cells (hNSPCs), but can also be used for many other cell types.

Knowledge of the exact number of viable cells is required for many tissue culture manipulations. This protocol describes how to differentiate between live and dead cells and quantify cells using a hemacytometer. Although it describes counting human neural stem/precursor cells (hNSPCs), it can be used for other cell types.

ספירת אדם בתאי גזע עצביים

Knowledge of the exact number of viable cells in a given volume of a cell suspension is required for many routine tissue culture manipulations, such as ...

Passaging Human Neural Stem Cells

The ability to manipulate human neural stem/precursor cells (hNSPCs) in vitro provides a means to investigate their utility as cell transplants for therapeutic purposes as well as to explore many fundamental processes of human neural development and pathology.  This protocol presents a simple method of culturing and passaging hNSPCs in hopes of standardizing this technique and increasing reproducibility of human stem cell research.  The hNSPCs we use were isolated from cadaveric postnatal brain cortices by the National Human Neural Stem Cell Resource and grown as adherent cultures on flasks coated with fibronectin (Palmer et al., 2001; Schwartz et al., 2003).  We culture our hNSPCs in a DMEM:F12 serum-free media supplemented with EGF, FGF, and PDGF and passage them 1:2 approximately every seven days.  Using these conditions, the majority of the cells in the culture maintain a bipolar morphology and express markers of undifferentiated neural stem cells (such as nestin and sox2).

The ability to manipulate human neural stem/precursor cells (hNSPCs) in vitro allows to investigate their utility as cell transplants for therapeutic purposes and to explore human neural development. This protocol presents a method of culturing and passaging hNSPCs in hopes of increasing reproducibility of human stem cell research.

Passaging אדם בתאי גזע עצביים

The ability to manipulate human neural stem/precursor cells (hNSPCs) in vitro provides a means to investigate their utility as cell transplants for ...

Immunocytochemistry: Human Neural Stem Cells

Immunocytochemistry is a very powerful and fairly straightforward method for determining the presence, subcellular localization, and relative abundance of an antigen of interest, most commonly a protein, in cultured cells. This protocol presents an easy-to-follow series of steps that will enable researchers to conserve primary and secondary antibodies while getting high quality, reproducible qualitative and quantitative data out of their staining. There are two aspects of this protocol that help to conserve the volume of antibody necessary for staining. For one, the cells are grown on small, circular coverslips that are placed in wells of a tissue culture plate. After fixation, the cells on coverslips can be removed from the wells of the plate. For antibody staining, the coverslip with cells is inverted onto a small drop of antibody solution on parafilm and is covered with a second piece of parafilm to prevent drying. Using this method, only ~25 &mu;l of antibody solution is needed for each coverslip (or sample) to be stained. This protocol describes immunostaining of human neural stem/precursor cells (hNSPCs), but can be used for many other cell types.

Immunocytochemistry is a powerful method to determine the presence, subcellular localization, and relative abundance of an antigen of interest in cultured cells. This protocol presents an easy-to-follow series of steps that will enable one to conserve antibodies and get the most out of one's staining.

Immunocytochemistry: אדם בתאי גזע עצביים

Immunocytochemistry is a very powerful and fairly straightforward method for determining the presence, subcellular localization, and relative abundance of ...

Differentiation of Embryonic Stem Cells into Oligodendrocyte Precursors

Oligodendrocytes are the myelinating cells of the central nervous system. For regenerative cell therapy in demyelinating diseases, there is significant interest in deriving a pure population of lineage-committed oligodendrocyte precursor cells (OPCs) for transplantation. OPCs are characterized by the activity of the transcription factor Olig2 and surface expression of a proteoglycan NG2. Using the GFP-Olig2 (G-Olig2) mouse embryonic stem cell (mESC) reporter line, we optimized conditions for the differentiation of mESCs into GFP+Olig2+NG2+ OPCs. In our protocol, we first describe the generation of embryoid bodies (EBs) from mESCs. Second, we describe treatment of mESC-derived EBs with small molecules: (1) retinoic acid (RA) and (2) a sonic hedgehog (Shh) agonist purmorphamine (Pur) under defined culture conditions to direct EB differentiation into the oligodendroglial lineage. By this approach, OPCs can be obtained with high efficiency (&gt;80%) in a time period of 30 days. Cells derived from mESCs in this protocol are phenotypically similar to OPCs derived from primary tissue culture. The mESC-derived OPCs do not show the spiking property described for a subpopulation of brain OPCs in situ. To study this electrophysiological property, we describe the generation of spiking mESC-derived OPCs by ectopically expressing NaV1.2 subunit. The spiking and nonspiking cells obtained from this protocol will help advance functional studies on the two subpopulations of OPCs.

We describe a small molecule-based protocol for differentiation of mouse embryonic stem cells into oligodendrocyte precursor cells (OPCs). This protocol generates Olig2+NG2+ OPCs with high efficiency by 30 days of differentiation. We also describe a method to generate "spiking" OPCs that can fire action potentials.

התמיינות של תאים עובריים לתוך מבשרי Oligodendrocyte

Oligodendrocytes are the myelinating cells of the central nervous system. For regenerative cell therapy in demyelinating diseases, there is significant ...

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 ...

Reprogramming Human Somatic Cells into Induced Pluripotent Stem Cells (iPSCs) Using Retroviral Vector with GFP

Human embryonic stem cells (hESCs) are pluripotent and an invaluable cellular sources for in vitro disease modeling and regenerative medicine1. It has been previously shown that human somatic cells can be reprogrammed to pluripotency by ectopic expression of four transcription factors (Oct4, Sox2, Klf4 and Myc) and become induced pluripotent stem cells (iPSCs)2-4 . Like hESCs, human iPSCs are pluripotent and a potential source for autologous cells. Here we describe the protocol to reprogram human fibroblast cells with the four reprogramming factors cloned into GFP-containing retroviral backbone4. Using the following protocol, we generate human iPSCs in 3-4 weeks under human ESC culture condition. Human iPSC colonies closely resemble hESCs in morphology and display the loss of GFP fluorescence as a result of retroviral transgene silencing. iPSC colonies isolated mechanically under a fluorescence microscope behave in a similar fashion as hESCs. In these cells, we detect the expression of multiple pluripotency genes and surface markers.

Reprogramming Human Somatic Cells into Induced Pluripotent Stem Cells iPSCs Using Retroviral Vector with GFP

A method to generate human induced pluripotent stem cells (iPSCs) via retrovirus-mediated ectopic expression of OCT4, SOX2, KLF4 and MYC is described. A practical way to identify human iPSC colonies based on GFP expression is also discussed.

תכנות מחדש האדם תאים סומטיים תוך המושרה בתאי גזע pluripotent (iPSCs) באמצעות וקטור retroviral עם GFP

Human embryonic stem cells (hESCs) are pluripotent and an invaluable cellular sources for in vitro disease modeling and regenerative medicine1. It has ...

Differentiation of Newborn Mouse Skin Derived Stem Cells into Germ-like Cells In vitro

Studying germ cell formation and differentiation has traditionally been very difficult due to low cell numbers and their location deep within developing embryos. The availability of a "closed" in vitro based system could prove invaluable for our understanding of gametogenesis. The formation of oocyte-like cells (OLCs) from somatic stem cells, isolated from newborn mouse skin, has been demonstrated and can be visualized in this video protocol. The resulting OLCs express various markers consistent with oocytes such as Oct4 , Vasa , Bmp15, and Scp3. However, they remain unable to undergo maturation or fertilization due to a failure to complete meiosis. This protocol will provide a system that is useful for studying the early stage formation and differentiation of germ cells into more mature gametes. During early differentiation the number of cells expressing Oct4 (potential germ-like cells) reaches ~5%, however currently the formation of OLCs remains relatively inefficient. The protocol is relatively straight forward though special care should be taken to ensure the starting cell population is healthy and at an early passage.

Differentiation of Newborn Mouse Skin Derived Stem Cells into Germ-like Cells In vitro

In recent years the formation of germ cells using in vitro culture methods has been demonstrated using several different types of somatic stem cells. This article will visually present the differentiation of newborn mouse derived stem cells to early oocyte-like cells.

התמיינות של תאי העור של עכבר יילוד נגזר גזע לתאים דמוי נבט<em&gt; במבחנה</em

Studying  germ cell formation and differentiation has traditionally been very difficult  due to low cell numbers and their location deep within developing ...

Generation of Transgenic Hydra by Embryo Microinjection

As a member of the phylum Cnidaria, the sister group to all bilaterians, Hydra can shed light on fundamental biological processes shared among multicellular animals. Hydra is used as a model for the study of regeneration, pattern formation, and stem cells. However, research efforts have been hampered by lack of a reliable method for gene perturbations to study molecular function. The development of transgenic methods has revitalized the study of Hydra biology1. Transgenic Hydra allow for the tracking of live cells, sorting to yield pure cell populations for biochemical analysis, manipulation of gene function by knockdown and over-expression, and analysis of promoter function. Plasmid DNA injected into early stage embryos randomly integrates into the genome early in development. This results in hatchlings that express transgenes in patches of tissue in one or more of the three lineages (ectodermal epithelial, endodermal epithelial, or interstitial). The success rate of obtaining a hatchling with transgenic tissue is between 10% and 20%. Asexual propagation of the transgenic hatchling is used to establish a uniformly transgenic line in a particular lineage. Generating transgenic Hydra is surprisingly simple and robust, and here we describe a protocol that can be easily implemented at low cost.

Generation of Transgenic Hydra by Embryo Microinjection

Stably transgenic Hydra are made by microinjection of plasmid DNA into embryos followed by random genomic integration and asexual propagation to establish a uniform line. Transgenic Hydra are used to track cell movements, overexpress genes, study promoter function, or knock down gene expression using RNAi.

דור של הטרנסגניים<em&gt; הידרה</em&gt; על ידי Microinjection העובר

As a member of the phylum Cnidaria, the sister group to all bilaterians, Hydra can shed light on fundamental biological processes shared among ...

Generation and Maintenance of Primate Induced Pluripotent Stem Cells Derived from Urine

Cross-species approaches studying primate pluripotent stem cells and their derivatives are crucial to better understand the molecular and cellular mechanisms of disease, development, and evolution. To make primate induced pluripotent stem cells (iPSCs) more accessible, this paper presents a non-invasive method to generate human and non-human primate iPSCs from urine-derived cells, and their maintenance using a feeder-free culturing method.
The urine can be sampled from a non-sterile environment (e.g., the cage of the animal) and treated with a broad-spectrum antibiotic cocktail during primary cell culture to reduce contamination efficiently. After propagation of the urine-derived cells, iPSCs are generated by a modified transduction method of a commercially available Sendai virus vector system. First&#160;iPSC colonies may already be visible after 5 days, and can be picked after 10 days at the earliest. Routine clump passaging with enzyme-free dissociation buffer supports pluripotency of the generated iPSCs for more than 50 passages.

The present protocol describes a method to isolate, expand, and reprogram human and non-human primate urine-derived cells to induced pluripotent stem cells (iPSCs), as well as instructions for feeder-free maintenance of the newly generated iPSCs.